U.S. patent number 7,790,207 [Application Number 10/465,665] was granted by the patent office on 2010-09-07 for colour reduction in canola protein isolate.
This patent grant is currently assigned to Burcon Nutrascience (MB) Corp.. Invention is credited to Brent E. Green, Radka Milanova, Kevin I. Segall, Lei Xu.
United States Patent |
7,790,207 |
Green , et al. |
September 7, 2010 |
Colour reduction in canola protein isolate
Abstract
In the recovery of canola protein isolates from canola oil seeds
steps are taken to inhibit the formation of coloring components and
to reduce the presence of materials tending to form coloring
components, to obtain a lighter and less yellow canola protein
isolate.
Inventors: |
Green; Brent E. (Winnipeg,
CA), Xu; Lei (Ottawa, CA), Milanova;
Radka (Vancouver, CA), Segall; Kevin I.
(Winnipeg, CA) |
Assignee: |
Burcon Nutrascience (MB) Corp.
(Winnipeg, Manitoba, CA)
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Family
ID: |
30003131 |
Appl.
No.: |
10/465,665 |
Filed: |
June 20, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040077838 A1 |
Apr 22, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60389957 |
Jun 20, 2002 |
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60423985 |
Nov 6, 2002 |
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Current U.S.
Class: |
424/776; 424/400;
424/725; 424/439 |
Current CPC
Class: |
A23J
3/14 (20130101); A23L 5/49 (20160801); C07K
14/415 (20130101); A23J 1/142 (20130101); A23J
1/14 (20130101); A23L 5/40 (20160801) |
Current International
Class: |
A61K
36/00 (20060101); A61K 47/00 (20060101); A61K
9/00 (20090101); A01N 65/00 (20060101) |
Field of
Search: |
;530/370
;424/724,400,439,442 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 322 462 |
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Nov 1974 |
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DE |
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247 835 |
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Jul 1987 |
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DE |
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100 35 292 |
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Feb 2002 |
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DE |
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Other References
Murray et al. "Rapeseed: a potential global source of high quality
plant protein" Apr. 2001 pp. 30-34, XP002207606. cited by other
.
Database WPI, Section Ch, Derwent Publication Ltd., London, GB;
XP002246020 & JP 05 04597 A. (Feb. 21, 1993). cited by other
.
Krzyzaniak A. et al., The Structure and properties of Napin-Seed
Storage Protein from Rape vol. 42, No. 3/4, 1998, pp. 201-204.
XP000861830. cited by other.
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Primary Examiner: Tate; Christopher R
Assistant Examiner: Winston; Randall
Attorney, Agent or Firm: Sim & McBurney Stewart; Michael
I.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application claims priority pursuant to 35 USC 119(e) from
copending U.S. Provisional Patents Applications Nos. 60/389,957
filed Jun. 20, 2002 and 60/432,985 filed Nov. 6, 2002.
Claims
What we claim is:
1. A process of forming a canola protein isolate, comprising:
processing canola seeds to form a canola protein meal, extracting
the canola protein meal to form an aqueous canola protein solution,
concentrating the aqueous canola protein solution to form a
concentrated aqueous canola protein solution, recovering a canola
protein isolate from said concentrated aqueous canola protein
solution by adding the concentrated aqueous solution to chilled
water to form a protein micellar mass, and separating the protein
micellar mass from supernatant, processing said supernatant to
recover additional canola protein isolate therefrom by
concentrating the supernatant, subjecting the concentrated
supernatant to diafiltration and recovering canola protein isolate
from the diafiltered supernatant, wherein at least one process step
during said process results in a canola protein isolate having a
decreased color.
2. The process of claim 1, wherein said processing of canola seeds
comprises inactivation of myrosinase in the canola oil seeds.
3. The process of claim 1, wherein said extraction step is effected
on low temperature desolventized meal or air desolventized
meal.
4. The process of claim 1, wherein said concentrated aqueous canola
protein solution is subjected to diafilteration prior to adding to
chilled water.
5. The process of claim 1, wherein said at least one process step
comprises solvent extracting said canola protein meal with an
alcohol.
6. The process of claim 1 wherein said at least one process step
comprises solvent extracting said canola protein isolate with an
aqueous alcoholic solution.
Description
FIELD OF INVENTION
The present invention relates to the recovery of canola protein
isolate from canola seed meals.
BACKGROUND TO THE INVENTION
In U.S. Pat. Nos. 5,844,086 and 6,005,076 ("Murray II"), assigned
to the assignee hereof and the disclosures of which are
incorporated herein by reference, there is described a process for
the isolation of protein isolates from oil seed meal having a
significant fat content, including canola oil seed meal having such
content. The steps involved in this process include solubilizing
proteinaceous material from oil seed meal, which also solubilizes
fat in the meal, and removing fat from the resulting aqueous
protein solution. The aqueous protein solution may be separated
from the residual oil seed meal before or after the fat removal
step. The defatted protein solution then is concentrated to
increase the protein concentration while maintaining the ionic
strength substantially constant, after which the concentrated
protein solution may be subjected to a further fat removal step.
The concentrated protein solution then is diluted to cause the
formation of a cloud-like mass of highly associated protein
molecules as discrete protein droplets in micellar form. The
protein micelles are allowed to settle to form an aggregated,
coalesced, dense amorphous, sticky gluten-like protein isolate
mass, termed "protein micellar mass" or PMM, which is separated
from residual aqueous phase and dried.
The protein isolate has a protein content (as determined by
Kjeldahl nitrogen or other convenient procedure N.times.6.25) of at
least about 90 wt %, is substantially undenatured (as determined by
differential scanning calorimetry) and has a low residual fat
content. The term "protein content" as used herein refers to the
quantity of protein in the protein isolate expressed on a dry
weight basis. The yield of protein isolate obtained using this
procedure, in terms of the proportion of protein extracted from the
oil seed meal which is recovered as dried protein isolate was
generally less than 40 wt %, typically around 20 wt %.
The procedure described in the aforementioned Murray II patent was
developed as a modification to and improvement on the procedure for
forming a protein isolate from a variety of protein source
materials, including oil seeds, as described in U.S. Pat. No.
4,208,323 (Murray IB). The oil seed meals available in 1980, when
U.S. Pat. No. 4,208,323 issued, did not have the fat contamination
levels of the canola oil seed meals available at the time of the
Murray II patents, and, as a consequence, the procedure of the
Murray IB patent cannot produce from such oil seed meals,
proteinaceous materials which have more than 90 wt % protein
content. There is no description of any specific experiments in the
Murray IB patent carried out using rapeseed (canola) meal as the
starting material.
The Murray IB patent, itself was designed to be an improvement on
the process described in U.S. Pat. Nos. 4,169,090 and 4,285,862
(Murray IA) by the introduction of the concentration step prior to
dilution to form the PMM. The Murray IA patents describe one
experiment involving rapeseed but provides no indication of the
purity of the product. The concentration step described in the
Murray IB patent served to improve the yield of protein isolate
from around 20% for the Murray IA process.
One difficulty which the canola protein isolates produced by such
prior procedures possess is a relatively dark yellow colour and an
undesirable flavour. Phenolic compounds have been reported to be
responsible for these problems of canola protein products including
meal. Canola contains about ten times the quantity of phenolic
compounds as is found in soybeans and may comprise sinapine and
condensed tannins. Upon oxidation, phenolic compounds can give rise
to the development of a dark colour. This problem is particularly
acute with canola protein products produced by isoelectric
precipitation where the strongly alkaline conditions lead to ready
oxidation of phenolic compounds to quinones, which then react with
the protein and impart a dark green or brown colour to the protein
and solutions thereof. Other compounds and reactions also may
contribute to colour formation.
SUMMARY OF INVENTION
The applicants provide herein an improvement in a process of
forming a canola protein isolate wherein canola seeds are processed
to form a canola protein meal, the canola protein meal is extracted
to form an aqueous protein solution, the aqueous protein solution
is concentrated, and the canola protein isolate is recovered from
the concentrated aqueous protein solution.
Phenolic compounds are extracted from the canola meal in the
extraction step and the quantity of free phenolics present can be
extracted by UV absorbance at 330 nm (A330). Such phenolics are
prone to oxidation to quinones and which react with proteins to
form coloured compounds, which tend to absorb at higher
wavelengths. Determination of absorbance at 420 nm (A420) provides
a more direct measurement of actual visual yellow colouration of
the isolate and canola protein solutions. In the present invention,
during the processing to obtain the canola protein isolate, steps
are taken to remove the phenolics so that they are unable to form
visible colouring components, to inhibit oxidation of phenolics to
visible colouring components and to remove other visible colouring
components.
The improvement provided by one aspect of the present invention
involves effecting at least one process step during the
above-described process which results in a canola protein isolate
having a decreased colour. The applicants have taken a multifaceted
approach to this procedure and one or more of several steps may
taken including: processing of canola seed treatment of meal
utilizing a specific form of canola protein meal effecting
extraction of a canola protein under specific conditions processing
of extract processing of the recovered canola protein isolate
Two or more of such procedures may be employed and often
combinations of such procedures are used.
Where the processing of seeds is effected, the procedure includes
at least inactivation of myrosinase in the seeds while still
hulled. By inactivating the myrosinase, any catalytic effect of the
myrosinase on the breakdown of glucosinolates into the sulfur
components which are anti-nutrients that contribute to taste and
colour. The procedure is more fully described in copending U.S.
patent application Ser. No. 10/871,065 filed Jun. 21, 2004,
assigned to the assignee hereof and the disclosure of which is
incorporated herein by reference.
The treatment of meal may involve extraction of the meal with a
water miscible organic solvent including alcohols, such as ethanol,
to extract phenolics and/or other colouring components.
Where a specific form of canola protein meal is used, such meal may
be an air-desolventized meal, prepared by removing residual solvent
from solvent extraction of canola oil seed meal at a temperature
below about 50.degree. C., generally at an ambient temperature of
about 15.degree. to about 30.degree. C.
In addition, the specific form of canola protein meal may be a
low-temperature toasted canola oil seed meal, prepared by removing
residual solvent from solvent extraction of canola oil seed meal at
an elevated temperature below about 100.degree. C.
Where the process step involves the extraction step, the extraction
step may be effected in the presence of an antioxidant to inhibit
oxidation of phenolics and visible colour formation. Alternatively
or in combination, the aqueous protein solution formed by the
extraction step may be treated with at least one colouring
component adsorbing agent. In addition, or alternatively, the
treatment with at least one colouring component adsorbing agent may
be effected on the concentrated canola protein solution formed in
the concentration step.
Where the process step involves the concentration step, the
concentrated aqueous canola protein solution is subject to
diafiltration to wash colourants from the concentrated canola
protein solution. The diafiltration may be carried out using an
aqueous solution containing an antioxidant to inhibit oxidation of
phenolics and visible colour formation during the
diafiltration.
Where the process step involves the recovered canola protein
isolate, the process step may involve extraction of the canola
protein isolate using aqueous alcoholic solutions, such as aqueous
ethanol, to extract phenolics and/or visible colourants from the
canola protein isolate.
The canola protein isolate may be recovered from the concentrated
aqueous protein solution by adding the concentrated aqueous
solution to chilled water to form a protein micellar mass, and
separating the protein micellar mass from supernatant.
The supernatant may be processed to recover additional canola
protein isolate therefrom by concentrating the supernatant,
subjecting the concentrated supernatant to diafiltration to remove
phenolics and/or visible colorants from the concentrated
supernatant and then recovering the canola protein isolate from the
diafiltered supernatant, such as by drying the diafiltered
supernatant.
By preventing colour formation and by improving the colour of the
canola protein isolate, the product may be used in a wider range of
applications. The removal and prevention of the formation of
colourants in accordance with this invention is thought also to
improve the flavour of the canola protein isolates.
The protein isolate produced according to the process herein may be
used in conventional applications of protein isolates, such as,
protein fortification of processed foods, emulsification of oils,
body formers in baked goods and foaming agents in products which
entrap gases. In addition, the protein isolate may be formed into
protein fibers, useful in meat analogs, may be used as an egg white
substitute or extender in food products where egg white is used as
a binder. The canola protein isolate may be used as nutritional
supplements. Other uses of the canola protein isolate are in pet
foods, animal feed and in industrial and cosmetic applications and
in personal care products.
In accordance with one specific aspect of the present invention,
there is provided a process of preparing a canola protein isolate
from canola oil seed meal, which comprises (a) extracting the
canola oil seed meal and to cause solubilization of the protein in
the canola oil seed meal to form an aqueous protein solution having
a pH of about 5 to about 6.8 by using an aqueous salt solution
containing an antioxidant, (b) separating the aqueous protein
solution from residual oil seed meal, (c) increasing the protein
concentration of said aqueous protein solution while maintaining
the ionic strength substantially constant by use of a selective
membrane technique to provide a concentrated protein solution, (d)
diluting said concentrated protein solution into chilled water
having a temperature of below about 15.degree. C. to cause the
formation of discrete protein micelles in the aqueous phase, (e)
settling the protein micelles to form an amorphous, sticky,
gelatinous, gluten-like protein micellar mass, and (f) recovering
the protein micellar mass from supernatant, the protein micellar
mass having a protein content of at least about 90 wt %
(N.times.6.25) on a dry weight basis.
In accordance with another specific aspect of the present
invention, there is provided a process of preparing a canola
protein solution from canola oil seed meal, which comprises (a)
washing said canola oil seed meal with an alcohol, (b) extracting
the washed canola oil seed meal to cause solubilization of the
protein in the washed canola oil seed meal to form an aqueous
protein solution having a pH of about 5 to about 6.8, (c)
separating the aqueous protein solution from residual oil seed
meal, (d) increasing the protein concentration of said aqueous
protein solution while maintaining the ionic straight substantially
constant by use of a selective membrane technique to provide a
concentrated protein solution, (e) diluting said concentrated
protein solution into chilled water having a temperature of below
about 15.degree. C. to cause the formation of discrete protein
micelles in the aqueous phase, (f) settling the protein micelles to
form an amorphous, sticky, gelatinous, gluten-like protein micellar
mass, and (g) recovering the protein micellar mass from
supernatant, the protein micellar mass having a protein content of
at least about 90 wt % (N.times.6.25) on a dry weight basis.
In accordance with a further specific aspect of the present
invention, three s provided a process of preparing a canola protein
isolate from canola oil seed meal, which comprises (a) extracting
the canola oil seed meal to cause solubilization of the protein in
the canola oil seed meal to form an aqueous protein solution having
a pH about 5 to about 6.8, (b) separating the aqueous protein
solution from residual oil seed meal, (c) increasing the protein
concentration of said aqueous protein solution while maintaining
the ionic strength substantially constant by effecting
ultrafiltration of the aqueous protein solution to provide a
concentrated protein solution, (d) subjecting the concentrated
protein solution to diafiltration, (e) diluting the diafiltered
protein solution into chilled water having a temperature below
about 15.degree. C. to cause the formation of discrete protein
micelles in the aqueous phase, (f) settling the protein micelles to
form an amorphous, sticky, gelatinous, gluten-like protein micellar
mass, and (g) recovering the protein micellar mass from
supernatant, the protein micellar mass having a protein content of
at least about 90 wt % (N.times.6.25) on a dry weight basis.
In accordance with a yet further aspect of the present invention,
there s provided a process of preparing a canola protein isolate
from canola oil seed meal, which comprises (a) extracting the
canola oil seed meal to cause solubilization of the protein in the
canola oil seed meal to form an aqueous protein solution having a
pH of about 5 to about 6.8, (b) separating the aqueous protein
solution from the residual oil seed meal, (c) increasing the
protein concentration of said aqueous protein solution while
maintaining the ionic strength substantially constant by use of a
selective membrane technique to form a concentrated protein
solution, (d) diluting said concentrated protein solution into
chilled water having a temperature below about 15.degree. C. to
cause the formation of discrete protein micelles in the aqueous
phase, (e) settling the protein micelles to form an amorphous,
sticky, gelatinous, gluten-like protein micellar mass, (f)
separating the protein micellar mass from supernatant, (g) drying
the protein micellar mass to provide a canola protein isolate
having a protein content of at least about 90 wt % (N.times.6.25)
on a dry weight basis, and (h) extracting said canola protein
isolate with an aqueous alcoholic solution.
In accordance with an additional aspect of the present invention,
three is provided a process of preparing a canola protein isolate
from canola oil seed meal, which comprises (a) extracting the
canola oil seed meal to cause solubilization of the protein in the
canola oil seed meal to form an aqueous protein solution having a
pH of about 5 to about 6.8, (b) separating the aqueous protein
solution from residual oil seed meal, (c) increasing the protein
concentration of said aqueous protein solution while maintaining
the ionic strength substantially constant by use of a selective
membrane technique to provide a concentrated protein solution, (d)
pasteurizing the concentrated protein solution to form a
pasteurized protein solution, (e) diluting the pasteurized protein
solution into chilled water having a temperature below about
15.degree. C. to cause the formation of discrete protein micelles
in the aqueous phase, (f) settling the protein micelles to form an
amorphous, sticky, gelatinous, gluten-like protein micellar mass,
and (g) recovering the protein micellar mass from supernatant, the
protein micellar mass having a protein content of at least about 90
wt % (N.times.6.25) on a dry weight basis.
In accordance with another aspect of the present invention, there
is provided a process of preparing a canola protein isolate from
canola oil seed, which comprises (a) treating canola oil seeds to
inactivate myrosinases contained in the oil seeds to produce
treated oil seeds, (b) processing said oil seeds to remove canola
oil therefrom and produce a canola oil seed meal, (c) extracting
the canola oil seed to cause solubilization of the protein in the
canola oil seed to form an aqueous solution having a pH of about 5
to about 6.8, (d) separating the aqueous protein solution from
residual oil seed meal, (e) increasing the protein concentration of
said aqueous protein solution which maintaining the ionic strength
substantially constant by use of a selective membrane technique to
provide a concentrated protein solution, (f) diluting the
concentrated protein solution into chilled water having a
temperature below about 15.degree. C. to cause formation of
discrete protein micelles in the aqueous phase, (g) settling the
protein micelles to form an amorphous, sticky, gelatinous,
gluten-like protein micellar mass, and (h) recovering the protein
micellar mass from supernatant, the protein micellar mass having a
protein content of at least about 90 wt % of (N.times.6.25) on a
dry weight basis.
Canola is also known as rapeseed or oil seed rape.
GENERAL DESCRIPTION OF INVENTION
Colour improvement may be achieved by the processing of seeds.
Hulled seeds are subjected to heat inactivation of myrosinase using
steam. The inactivated seeds then may be processed in conventional
manner to recover oil from the seeds and to form canola oil seed
meal.
It is preferred, in accordance with one embodiment of the
invention, for the oil seed meal to be desolventized by toasting at
an elevated temperature below about 100.degree. C., since such meal
gives rise to less colour development than meal desolventized using
conventional, much higher, toasting temperatures. The formation of
a canola protein isolate having a protein content of at least about
90 wt % (N.times.6.25), preferably at least about 100 wt %, from
such meal is described in copending U.S. patent application Ser.
No. 10/314,202 filed Dec. 9, 2002, assigned to the assignee hereof
and the disclosure of which is incorporated herein by
reference.
More preferably, the oil seed meal is desolventized in air at
temperatures below about 50.degree. C., preferably around ambient
temperature about 15.degree. to about 30.degree. C., since even
less colour than in the case of the use of the toasted meal is
present in the extract solution. The formation of a canola protein
isolate having a protein content of at least about 90 wt %
(N.times.6.25), preferably at least about 100 wt %, from such meal
is described in copending U.S. applications Nos. 60/390,126 filed
Jun. 21, 2002 and 60/401,712 filed Aug. 8, 2002 and Ser. No.
10/465,238 filed Jun. 20, 2003, assigned to the assignee hereof and
the disclosures of which are incorporated herein by reference.
Canola protein isolates can be formed from canola oil seed meal. In
copending U.S. Patent Applications Nos. 60/288,415 filed May 4,
2001, 60/326,987 filed Oct. 5, 2001, 60/331,066 filed Nov. 7, 2001,
60/333,494 filed Nov. 26, 2001, 60/374,801 filed Apr. 24, 2002 and
Ser. No. 10/137,391 filed May 3, 2002 (WO 02/089597), all assigned
to the assignee hereof and the disclosures of which are
incorporated herein by reference, there is described a method of
making canola protein isolates from canola oil seed meal, such
isolates having at least about 100 wt % protein content
(N.times.6.25). The procedure involves a multiple step process
comprising extracting canola oil seed meal using a salt solution,
separating the resulting aqueous protein solution from residual oil
seed meal, increasing the protein concentration of the aqueous
solution to at least about 200 g/L while maintaining the ionic
strength substantially constant by using a selective membrane
technique, diluting the resulting concentrated protein solution
into chilled water to cause the formation of protein micelles,
settling the protein micelles to form an amorphous, sticky,
gelatinous gluten-like protein micellar mass (PMM), and recovering
the protein micellar mass from supernatant having a protein content
of at least about 100 wt % (N.times.6.25). As used herein, protein
content is determined on a dry weight basis. The recovered PMM may
be dried.
In one embodiment of the process described above and as
specifically described in U.S. Patent Applications Nos. 60/326,987,
60/331,066, 60/333,494, 60/374,801 and Ser. No. 10/137,391, the
supernatant from the PMM settling step is processed to recover a
protein isolate comprising dried protein isolate from the wet PMM
and supernatant. This procedure may be effected by initially
concentrating the supernatant using ultrafiltration membranes,
mixing the concentrated supernatant with the wet PMM and drying the
mixture. The resulting canola protein isolate has a high purity of
at least about 90 wt % of protein (N.times.6.25), preferably at
least about 100 wt % protein (N.times.6.25).
In another embodiment of the process described above and as
specifically described in Applications Nos. 60/333,494, 60/374,801
and Ser. No. 10/137,391, the supernatant from the PMM settling step
is processed to recover a protein isolate from the supernatant.
This procedure may be effected by initially concentrating the
supernatant using ultrafiltration membranes and drying the
concentrate. The resulting canola protein isolate has a high purity
of at least about 90 wt % protein (N.times.6.25), preferably at
least about 100 wt % protein (N.times.6.25).
The procedures described in the aforementioned U.S. patent
applications are essentially batch procedures. In copending U.S.
Patent Applications Nos. 60/331,646 filed Nov. 20, 2001, 60/383,809
filed May 30, 2002 and Ser. No. 10/298,678 filed Nov. 19, 2002 (WO
03/043439), assigned to the assignee hereof and the disclosures of
which are incorporated herein by reference, there is described a
continuous process for making canola protein isolates. In
accordance therewith, canola oil seed meal is continuously mixed
with a salt solution, the mixture is conveyed through a pipe while
extracting protein from the canola oil seed meal to form an aqueous
protein solution, the aqueous protein solution is continuously
separated from residual canola oil seed meal, the aqueous protein
solution is continuously conveyed through a selective membrane
operation to increase the protein content of the aqueous protein
solution to at least about 200 g/L while maintaining the ionic
strength substantially constant, the resulting concentrated protein
solution is continuously mixed with chilled water to cause the
formation of protein micelles, and the protein micelles are
continuously permitted to settle while the supernatant is
continuously overflowed until the desired amount of PMM has
accumulated in the settling vessel. The PMM is removed from the
settling vessel and may be dried. The PMM has a protein content of
at least about 90 wt % (N.times.6.25), preferably at least about
100 wt % (N.times.6.25).
As described in the aforementioned U.S. Patent Applications Nos.
60/326,987, 60/331,066, 60/333,494, 60/333,494, 60/374,801 and Ser.
No. 10/137,391, the overflowed supernatant may be processed to
recover canola protein isolate therefrom.
In accordance with one embodiment of the present invention, the oil
seed meal may initially be solvent extracted to remove phenolics
and colourants therefrom. Such solvent extraction may be effected
using a water-soluble organic solvent for phenolics and/or visible
colourants, such as a water-soluble alcohol, preferably
ethanol.
The extraction may be effected by dispersing the canola oil seed
meal in the solvent at a w/v ratio of about 1:3 to about 1:10,
preferably about 1:5. The slurry may be stirred for about 5 to
about 60 minutes, preferably about 15 to about 30 minutes, at a
temperature of about 15.degree. to about 45.degree. C., preferably
about 30.degree. to about 35.degree. C. One suitable set of
conditions is a 30 minutes extraction at 35.degree. C. Such
extraction may be effected a multiple number of times until no
additional phenolics and/or visible colour are extracted.
In the process of the present invention, proteinaceous material is
solubilized from canola oil seed meal. The proteinaceous material
may be the protein naturally occurring in canola seed or the
proteinaceous material may have been modified by genetic
manipulation but possessing characteristic hydrophobic and polar
properties of the natural protein. The canola meal may be any
canola meal resulting from the removal of canola oil from canola
oil seed with varying levels of non-denatured protein, resulting,
for example, from hot hexane extraction or cold oil extrusion
methods. The processing of seed, when effected for the removal of
canola oil from canola oil seed usually is effected as a separate
operation from the protein isolate recovery procedure of the
present invention described herein.
Protein solubilization is effected most efficiently by using a food
grade salt solution since the presence of the salt enhances the
removal of soluble protein from the oil seed meal. Where the canola
protein isolate is intended for non-food uses, non-food-grade
chemicals may be used. The salt usually is sodium chloride,
although other salts, such as, potassium chloride, may be used. The
salt solution has an ionic strength of at least about 0.10,
preferably at least about 0.15, to enable solubilization of
significant quantities of protein to be effected. As the ionic
strength of the salt solution increases, the degree of
solubilization of protein in the oil seed meal initially increases
until a maximum value is achieved. Any subsequent increase in ionic
strength does not increase the total protein solubilized. The ionic
strength of the food grade salt solution which causes maximum
protein solubilization varies depending on the salt concerned and
the oil seed meal chosen. The food grade salt solution may have an
ionic strength ranging up to about 0.25.
In view of the greater degree of dilution required for protein
precipitation with increasing ionic strengths, it is usually
preferred to utilize an ionic strength value less than about 0.8,
and more preferably a value of about 0.15 to about 0.6.
In a batch process, the salt solubilization of the protein is
effected at a temperature of at least about 5.degree. C. and
preferably up to about 35.degree. C., preferably accompanied by
agitation to decrease the solubilization time, which is usually
about 10 to about 60 minutes. It is preferred to effect the
solubilization to extract substantially as much protein from the
oil seed meal as is practicable, so as to provide an overall high
product yield.
The lower temperature limit of about 5.degree. C. is chosen since
solubilization is impractically slow below this temperature while
the upper preferred temperature limit of about 35.degree. C. is
chosen since the process becomes uneconomic at higher temperature
levels in a batch mode.
In a continuous process, the extraction of the protein from the
canola oil seed meal is carried out in any manner consistent with
effecting a continuous extraction of protein from the canola oil
seed meal. In one embodiment, the canola oil seed meal is
continuously mixed with a food grade salt solution and the mixture
is conveyed through a pipe or conduit having a length and at a flow
rate for a residence time sufficient to effect the desired
extraction in accordance with the parameters described herein. In
such continuous procedure, the salt solubilization step is effected
rapidly, in a time of up to about 10 minutes, preferably to effect
solubilization to extract substantially as much protein from the
canola oil seed meal as is practicable. The solubilization in the
continuous procedure preferably is effected at elevated
temperatures, preferably above about 35.degree. C., generally up to
about 65.degree. C. or more.
The aqueous food grade salt solution and the canola oil seed meal
have a natural pH of about 5 to about 6.8 to enable a protein
isolate to be formed by the micellar route, as described in more
detail below.
At and close to the limits of the pH range, protein isolate
formation occurs only partly through the micelle route and in lower
yields than attainable elsewhere in the pH range. For these
reasons, mildly acidic pH values of about 5.3 to about 6.2 are
preferred.
The pH of the salt solution may be adjusted to any desired value
within the range of about 5 to about 6.8 for use in the extraction
step by the use of any convenient acid, usually hydrochloric acid,
or alkali, usually sodium hydroxide, as required.
The concentration of oil seed meal in the food grade salt solution
during the solubilization step may vary widely. Typical
concentration values are about 5 to about 15% w/v.
In accordance with one embodiment of the invention, an antioxidant
may be present in the food grade salt solution to inhibit oxidation
of phenols in the canola oil seed meal to components which react
with the protein and cause colour darkening. Any desired food-grade
antioxidant may be used, such as sodium sulfite and ascorbic acid.
The quantity of antioxidant employed in the aqueous food grade salt
solution depends on the material employed and may vary from about
0.01 to about 1 wt %, preferably about 0.05 to about 0.1 wt %.
Inhibition of oxidation of phenolics by the use of antioxidants
results in reduced extract colour (absorbance at 420 nm) while the
concentration of phenolics (absorbance at 330 nm) remains largely
unchanged.
In the presence of added sodium sulfite, even at a salt
concentration as low as 0.05 M, the protein concentration in the
extract at pH 6.3 was comparable with that with 0.15 M salt but
without sodium sulfite.
The protein solution resulting from the extraction step generally
has a protein concentration of about 5 to about 40 g/L, preferably
about 10 to about 30 g/L.
In choosing the parameters of the extraction step, the desire to
extract as much protein from the canola oil seed meal as is
possible is balanced with the desire to minimize the colour of the
resulting extract solution. In considering the data presented
herein, an extraction time of 30 minutes generally is sufficient to
extract all the protein that is going to be extracted under the
prevailing pH and salt molarity. A higher pH increases the amount
of protein extracted and results in a protein solution which is
visibly darker in colour (as measured by absorbance at A420).
It is possible to extract as much protein in 10 minutes with 0.1 M
saline at pH 8.0 as is extracted with 0.15 M saline at pH 6.3 in 30
minutes. There is a marked decrease in the A330 at extraction at pH
9.8 when compared to extraction with lower pH, although the colour
is visibly darker as the pH increases. An explanation for this
phenomenon may be that the phenolics are reacting to form yellow
colorants that do not absorb at A330, but rather absorb at higher
values between A360 and A400. For those reasons, extraction of the
canola protein meal is effected at a pH below 8.
The aqueous phase resulting from the extraction step then may be
separated from the residual canola meal, in any convenient manner,
such as by employing vacuum filtration, followed by centrifugation
and/or filtration to remove residual meal. The separated residual
meal may be dried for disposal.
Where the canola seed meal contains significant quantities of fat,
as described in the Murray II patents, then the defatting steps
described therein may be effected on the separated aqueous protein
solution and on the concentrated aqueous protein solution discussed
below.
As an alternative to extracting protein from the canola oil seed
meal with an aqueous salt solution, such extraction may be made
using water alone, although the utilization of water alone tends to
extract less protein from the canola oil seed meal than the aqueous
salt solution. Where such alternative is employed, then the salt,
in the concentrations discussed above, may be added to the protein
solution after separation from the residual oil seed meal in order
to maintain the protein in solution during the concentration step
described below. When a first fat removal step is carried out, the
salt generally is added after completion of such operation.
Another alternative procedure is to extract the canola oil seed
meal with a food grade salt solution at a relatively high pH value
above about 6.8, generally up to about 11. However, as noted above,
extraction at pH values greater than about 8 generally are avoided
since considerable visible colour formation results at such pH
values. The pH of the food grade salt solution, may be adjusted in
pH to the desired alkaline value by the use of any convenient
food-grade alkali, such as aqueous sodium hydroxide solution.
Alternatively, the protein may be extracted from the canola oil
seed meal with the salt solution at a relatively low pH below about
pH 5, generally down to about pH 3. Where such alternative is
employed, the aqueous phase resulting from the canola oil seed meal
extraction step then is separated from the residual canola meal, in
any convenient manner, such as by employing vacuum filtration,
followed by centrifugation and/or filtration to remove residual
meal. The separated residual canola meal may be dried for
disposal.
The aqueous protein solution resulting from the high or low pH
extraction step then may be pH adjusted to the range of about 5 to
about 6.8, preferably about 5.3 to about 6.2, as discussed above,
prior to further processing to recover canola protein isolate
mainly by the micelle route, as discussed below. Such pH adjustment
may be effected using any convenient acid, such as hydrochloric
acid, or alkali, such as sodium hydroxide, as appropriate.
Following extraction of the protein, the protein solution may be
subjected to one or more colour removal steps, in accordance with
another embodiment of the invention, including
ultrafiltration/diafiltration and contact with a colour adsorbing
agent. In the ultrafiltration step, the protein content of the
aqueous protein solution is increased while the salt concentration
remains unchanged. The ultrafiltration may be effected using
membranes having a molecular weight cut-off consistent with
permitting phenolics and colouring agents to pass through the
membrane with the permeate while the protein is retained, typically
an ultrafiltration membrane having a molecular weight cut-off of
about 3,000 to about 50,000 daltons, preferably about 5000 to about
10,000 daltons, having regard to differing membrane materials and
configurations. The membranes may be hollow-fibre membranes or
spiral-wound membranes. For continuous operation, the membranes may
be dimensioned to permit the desired degree of concentration as the
aqueous protein solution passes through the membranes.
The protein solution is concentrated by the ultrafiltration step
from about 4 to about 20 fold and preferably is effected to provide
a concentrated protein solution having a protein concentration of
at least about 200 g/L, more preferably at least about 250 g/L.
The concentrated protein solution then is subjected to a
diafiltration step using an aqueous salt solution of the same
molarity and pH as the extraction solution. Such diafiltration may
be effected using from about 2 to about 20 volumes of diafiltration
solution, preferably about 5 to about 10 volumes of diafiltration
solution. In the diafiltration operation, further quantities of
phenolics and visible colour are removed from the aqueous protein
solution by passage through the membrane with the permeate. The
diafiltration operation may be effected until no significant
further quantities of phenolics and visible colour are present in
the permeate. Such diafiltration may be effected using a membrane
having a molecular weight cut-off in the range of about 3000 to
about 50,000 daltons, preferably about 5,000 to about 10,000
daltons, having regard to different membrane materials and
configuration.
In accordance with an aspect of this embodiment of the invention,
an antioxidant may be present in the diafiltration medium using at
least part of the diaflitration step. The antioxidant may be any
convenient food grade antioxidant, such as sodium sulfite or
ascorbic acid. The quantity of antioxidant employed in the
diafiltration medium depends on the materials employed and may vary
from about 0.01 to about 1 wt %, preferably about 0.05 wt %. The
antioxidant serves to inhibit oxidation of phenolics present in the
concentrated canola protein isolate solution.
The concentration step and the diafiltration step may be effected
at any convenient temperature, generally about 20.degree. to about
60.degree. C., and for the period of time to effect the desired
degree of concentration. The temperature and other conditions used
to some degree depend upon the membrane equipment used to effect
the concentration and the desired protein concentration of the
solution.
In effecting the ultrafiltration/diafiltration operations, the
conditions are chosen having in mind the desire to provide a
protein solution having the highest protein concentration and
lowest colour. Based on the experiments reported below, the
ultrafiltration/diafiltration (UF/DF) procedure is able to
effectively reduce A330 values (phenolics concentration) by about
28% to about 74%, depending on pH and saline content. The
ultrafiltration/diafiltration operations also have the effect of
removing anti-nutritional factors, thereby improving the
nutritional quality of the canola protein isolate.
The UF/DF permeates had the highest A330 values at pH 8.0 and 6.3.
These high permeate A330 values are likely due to unbound phenolics
being able to pass through the membranes into the permeate while at
pH 9.8 and pH 11.0, the phenolics have reacted to form colourants
and do not absorb as strongly at A330.
Extractions effected at higher pH and saline level had the highest
starting A330 readings and in most cases the lowest final retentate
A330 readings. At the higher pH and saline values, permeates
contained higher levels of nitrogen, indicating protein loss.
A330 to protein ratios for final retentate indicate that the best
ratios are achieved at pH values from pH 6.3 and 8.0, indicating
that less A330 component per protein than the higher pH tests and a
more effective removal by the UF/DF procedures. In all but 0 M and
0.25 M saline concentrations, pH 6.3 had the best A330 to protein
ratio. Having regard thereto, pH 6.3 appears to be the best pH
level tested for diafiltering colour out of the protein solution
with 0.25 M saline being the best salt level for providing the
highest protein level.
The ultrafiltration/diafiltration operations may be followed by
treatment with a pigment adsorbing agent. In the aforementioned
copending U.S. Patent Applications Nos. 60/288,415, 60/326,987,
60/331,066, 60/333,494, 60/374,801 and Ser. No. 10/137,391 (WO
02/089597), there is described the use of powdered activated carbon
to effect colour reduction.
As described in such applications, such colour reduction step is
carried out on the canola protein solution prior to concentration
and results in a lighter colour and less intense yellow in the
product canola protein isolate compared to the absence of such
step. In accordance with another embodiment of the present
invention, the use of colour component adsorbing materials is
preferably effected on the concentrated and diafiltered canola
protein solution. Powdered activated carbon may be used herein as
well as granulated activated carbon (GAC). Another material which
may be used as a colour adsorbing agent is polyvinyl pyrrolidone.
Alternatively, in accordance with another embodiment of the
invention, the use of colour component adsorbing materials may be
effected on the canola protein solution prior to ultrafiltration
and optional diafiltration, and/or directly in the extraction step.
When the colour adsorbing material is employed prior to the
ultrafiltration step, diafiltration may be omitted, in the event
such diafiltration does not remove any additional phenolics and/or
visible colour.
In the experiments described below, polyvinyl pyrrolidone and GAC
reduced A330 values better at pH 6.3 and 8.0 than at pH 9.8 and 11,
probably due to binding of quinones to protein at the two higher pH
levels. Polyvinyl pyrrolidone produced a good reduction in A330
without protein loss. Other potential materials tested were
unsatisfactory, either as a result of unacceptable protein losses
or an inability to reduce the A330 of the solution.
The colour absorbing agent treatment step may be carried out under
any convenient conditions, generally at the ambient temperature of
the canola protein solution. For powdered activated carbon, an
amount of about 0.025% to about 5% w/v, preferably about 0.05% to
about 2% w/v, may be used. Where polyvinylpyrrolidone is used as
the colour adsorbing agent, an amount of about 0.5 to about 5 w/v,
preferably about 2 to about 3% w/v, may be used. The colour
adsorbing agent may be removed from the canola protein solution by
any convenient means, such as by filtration.
Following completion of the treatment by colour adsorbing agent on
diafiltered canola protein solution, the resulting protein solution
is processed to produce a canola protein isolate therefrom. The
recovery of the canola protein isolate may be effected in any
convenient manner, depending on the parameters of the protein
solution.
For example, the canola protein isolate may be recovered by
isoelectric precipitation from alkaline solutions or by a protein
micellar mass process from more neutral solutions. Alternatively,
the protein may be precipitated by increasing the salt
concentration.
The processing of the canola protein solution to recover a canola
protein isolate preferably is carried out using a protein micellar
mass process as described in the aforementioned U.S. patent
applications and in more detail below, since the extraction pH
conditions lead to less colour formation than those employed for
the isoelectric precipitation techniques.
Depending on the temperature employed in the colour removal steps
carried out on the aqueous canola protein solution, the
concentrated protein solution may be warmed to a temperature of at
least about 20.degree., and up to about 60.degree. C., preferably
about 25.degree. to about 40.degree. C., to decrease the viscosity
of the concentrated, optionally diafiltered, protein solution to
facilitate performance of the subsequent dilution step and micelle
formation. The concentrated and optionally diafiltered protein
solution should not be heated beyond a temperature above which the
temperature of the concentrated and optionally diafiltered protein
solution does not permit micelle formation on dilution by chilled
water. The concentrated and optionally diafiltered protein solution
may be subject to a further defatting operation, if required, as
described in the Murray II patents.
The concentrated protein solution resulting from the colour removal
steps may be subjected to pasteurization to kill any bacteria which
may have been present in the original meal as a result of storage
or otherwise and extracted from the meal into the canola protein
isolate solution in the extraction step. Such pasteurization may be
effected under any desired pasteurization conditions. Generally,
the concentrated and optionally diafiltered protein solution is
heated to a temperature of about 55.degree. to about 70.degree. C.,
preferably about 60.degree. to about 65.degree. C., for about 10 to
about 15 minutes, preferably about 10 minutes. The pasteurized
concentrated protein solution then may be cooled for further
processing as described below, preferably to a temperature of about
25.degree. to about 40.degree. C.
The concentrated protein solution resulting from the colour removal
steps and optional defatting and pasteurization steps then is
diluted to effect micelle formation by mixing the concentrated
protein solution with chilled water having the volume required to
achieve the degree of dilution desired. Depending on the proportion
of canola protein desired to be obtained by the micelle route and
the proportion from the supernatant, the degree of dilution of the
concentrated protein solution may be varied. With higher dilution
levels, in general, a greater proportion of the canola protein
remains in the aqueous phase.
When it is desired to provide the greatest proportion of the
protein by the micelle route, the concentrated protein solution is
diluted by about 15 fold or less, preferably about 10 fold or
less.
The chilled water with which the concentrated protein solution is
mixed has a temperature of less than about 15.degree. C., generally
about 3.degree. to about 15.degree. C., preferably less than about
10.degree. C., since improved yields of protein isolate in the form
of protein micellar mass are attained with these colder
temperatures at the dilution factors used.
In a batch operation, the batch of concentrated protein solution is
added to a static body of chilled water having the desired volume,
as discussed above. The dilution of the concentrated protein
solution and consequential decrease in ionic strength causes the
formation of a cloud-like mass of highly associated protein
molecules in the form of discrete protein droplets in micellar
form. In the batch procedure, the protein micelles are allowed to
settle in the body of chilled water to form an aggregated,
coalesced, dense, amorphous, sticky, gluten-like protein micellar
mass (PMM). The settling may be assisted, such as by
centrifugation. Such induced settling decreases the liquid content
of the protein micellar mass, thereby decreasing the moisture
content generally from about 70% by weight to about 95% by weight
to a value of generally about 50% by weight to about 80% by weight
of the total micellar mass. Decreasing the moisture content of the
micellar mass in this way also decreases the occluded salt content
of the micellar mass, and hence the salt content of dried
isolate.
Alternatively, the dilution operation may be carried out
continuously by continuously passing the concentrated protein
solution to one inlet of a T-shaped pipe, while the diluting water
is fed to the other inlet of the T-shaped pipe, permitting mixing
in the pipe. The diluting water is fed into the T-shaped pipe at a
rate sufficient to achieve the desired degree of dilution.
The mixing of the concentrated protein solution and the diluting
water in the pipe initiates the formation of protein micelles and
the mixture is continuously fed from the outlet from the T-shaped
pipe into a settling vessel, from which, when full, supernatant is
permitted to overflow. The mixture preferably is fed into the body
of liquid in the settling vessel in a manner which minimizes
turbulence within the body of liquid.
In the continuous procedure, the protein micelles are allowed to
settle in the settling vessel to form an aggregated, coalesced,
dense, amorphous, sticky, gluten-like protein micellar mass (PMM)
and the procedure is continued until a desired quantity of the PMM
has accumulated in the bottom of the settling vessel, whereupon the
accumulated PMM is removed from the settling vessel.
The combination of process parameters of concentrating the protein
solution to a protein content of at least about 200 g/L and the use
of a dilution factor less than about 15, result in higher yields,
often significantly higher yields, in terms of recovery of protein
in the form of protein micellar mass from the original meal
extract, and much purer isolates in terms of protein content than
achieved using any of the known prior art protein isolate forming
procedures discussed in the aforementioned US patents.
The settled isolate is separated from the residual aqueous phase or
supernatant, such as by decantation of the residual aqueous phase
from the settled mass or by centrifugation. The PMM may be used in
the wet form or may be dried, by any convenient technique, such as
spray drying, freeze drying or vacuum drum drying, to a dry form.
The dry PMM has a high protein content, in excess of about 90 wt %
protein, preferably at least about 100 wt % protein (calculated as
N.times.6.25), and is substantially undenatured (as determined by
differential scanning calorimetry). The dry PMM isolated from fatty
oil seed meal also has a low residual fat content, when the
procedures of the Murray II patents are employed, which may be
below about 1 wt %.
The supernatant from the PMM formation and settling step contains
significant amounts of canola protein, not precipitated in the
dilution step, and is processed to recover canola protein isolate
therefrom. The supernatant from the dilution step, following
removal of the PMM, is concentrated to increase the protein
concentration thereof. Such concentration is effected using any
convenient selective membrane technique, such as ultrafiltration,
using membranes with a suitable molecular weight cut-off permitting
low molecular weight species, including the salt and other
non-proteinaceous low molecular weight materials extracted from the
protein source material, to pass through the membrane, while
retaining canola protein in the solution. Ultrafiltration membranes
having a molecular weight cut-off of about 3000 to 10,000 daltons,
having regard to differing membrane materials and configuration,
may be used. Concentration of the supernatant in this way also
reduces the volume of liquid required to be dried to recover the
protein. The supernatant generally is concentrated to a protein
concentration of about 100 to about 400 g/L, preferably about 200
to about 300 g/L, prior to drying. Such concentration operation may
be carried out in a batch mode or in a continuous operation, as
described above for the protein solution concentration step.
In accordance with another embodiment of the invention, prior to
drying, the concentrated supernatant is subjected to a
diafiltration step using water. Such diafiltration may be effected
using about 2 to about 20 volumes of diafiltration solution,
preferably about 5 to about 10 volumes of diafiltration solution.
In the diafiltration operation, further quantities of phenolics and
visible colour are removed from the concentrated supernatant by
passage through the membrane with the permeate. The diafiltration
operation may be effected until no significant further quantities
of phenolics and visible colour are removed in the permeate. Such
diafiltration may be effected using a membrane having a molecular
weight cut-off in the range of about 3000 to about 50,000 daltons,
preferably about 5000 to about 10,000 daltons, having regard to
different membrane materials and configurations.
In accordance with an aspect of this embodiment of the invention,
an antioxidant may be present in the diafiltration medium. The
antioxidant may be any convenient food grade antioxidant, such as
sodium sulfite or ascorbic acid. The quantity of antioxidant
employed in the diafiltration medium depends on the materials
employed and may vary from about 0.01 to about 1 wt %, preferably
about 0.05 wt %. The antioxidant serves to inhibit oxidation of
phenolics present in the concentrated canola protein isolate
solution.
The concentrated supernatant may be used in the wet form or may be
dried by any convenient technique, such as spray drying, freeze
drying or vacuum drum drying, to a dry form to provide a further
canola protein isolate. Such further canola protein isolate has a
high protein content, in excess of about 90 wt %, preferably at
least about 100 wt % protein (calculated as N.times.6.25) and is
substantially undenatured (as determined by differential scanning
calorimetry).
If desired, at least a portion of the wet PMM may be combined with
at least a portion of the concentrated supernatant prior to drying
the combined protein streams by any convenient technique to provide
a combined canola protein isolate composition according to one
embodiment of the invention. The relative proportions of the
proteinaceous materials mixed together may be chosen to provide a
resulting canola protein isolate composition having a desired
profile of 2S/7S/12S proteins. Alternatively, the dried protein
isolates may be combined in any desired proportions to provide any
desired specific 2S/7S/12S protein profiles in the mixture. The
combined canola protein isolate composition has a high protein
content, in excess of about 90 wt %, preferably at least about 100
wt %, (calculated as N.times.6.25) and is substantially undenatured
(as determined by differential scanning calorimetry).
In another alternative procedure, where a portion only of the
concentrated supernatant is mixed with a part only of the PMM and
the resulting mixture dried, the remainder of the concentrated
supernatant may be dried as may any of the remainder of the PMM.
Further, dried PMM and dried supernatant also may be dry mixed in
any desired relative proportions, as discussed above.
By operating in this manner, a number of canola protein isolates
may be recovered, in the form of dried PMM, dried supernatant and
dried mixtures of various proportions by weight of PMM-derived
canola protein isolate and supernatant-derived canola protein
isolate, generally from about 5:95 to about 95:5 by weight, which
may be desirable for attaining differing functional and nutritional
properties based on the differing proportions of 2S/7S/12S proteins
in the compositions.
In accordance with another embodiment of the invention, PMM-derived
canola protein isolate and the supernatant-derived canola protein
isolate may be treated to remove colour-imparting components
thereof. Such treatment conveniently is effected using a
water-miscible organic solvent for phenolics and/or visible
colourants in mixture with water.
Since water-miscible organic solvent may be an alcohol. Preferred
is a blend of ethanol and water, generally in a volume ratio of
about 2:1 to about 1:2, preferably 1:1. The canola protein isolate
is dispersed in the solvent blend in an amount of about 5 to about
25% w/v, preferably about 8 to about 23% w/v, generally at ambient
temperature. The slurry of canola protein isolate may be mixed for
about 30 to about 60 minutes, preferably about 30 minutes.
Following the extraction period, the slurry is settled, such as by
centrifugation, and the canola protein isolate is recovered. The
extraction may be repeated, if desired, until no additional
phenolics and/or visible colourants are removed. The canola protein
isolate may be redispersed in an alcohol, such as ethanol, to
remove water from the isolate, which then may be separated and
dried.
EXAMPLES
Example 1
This Example shows the effect of various parameters on protein
extraction and protein solution colour.
A series of experimental runs was performed in which 37.5 g of
commercial canola meal (AL-016) was mixed with water containing
NaCl of desired concentration at the desired pH, at a meal
concentration of 7.5% w/v at 20.degree. C. Sodium chloride
concentrations employed were 0, 0.05, 0.10, 0.15 and 0.25 M and pH
values used were pH 6.3, 8.0, 9.8 and 11.0. A sample of about 30 mL
of extract was taken every 10 minutes during the 60 minutes
extraction period and centrifuged at 10,000.times.g for 5 minutes.
The supernatant of each sample was analyzed for protein
concentration at the end of the extraction period. The entire batch
was centrifuged at 10,000.times.g for fifteen minutes and the
supernatant was vacuum filtered using a 0.45 .mu.m micro filter.
The filtered supernatant was analyzed for protein content and for
free phenolics concentration (absorbance at 330 nm).
A 100 ml aliquot was drawn from the clarified supernatant for
ultrafiltration (UF) by a concentration factor of 4 using an Amicon
8400 unit with a membrane of 10,000 molecular weight cut-off. The
protein concentration and A330 absorbance of the 25 ml retentate
and pooled permeate were determined. The ultrafiltered solution was
subjected to diafiltration (DF) by a diavolume of 6, using 150 mL
of solution with the same salt concentration and same pH as used
for the extraction. At the end of the diafiltration both the
retentate and pooled permeate from the diafiltration were analyzed
for protein concentration and A330.
Aliquots of the final retentates were then passed through columns
containing one of five different adsorbents and again protein
concentration and A330 colour were tested on the resulting protein
solutions. The adsorbents were Amberlite XAD-16 HP (polymeric
absorbent), Amberlite SF120NA (a cation exchanger), Polyclar Super
R (polyvinyl pyrrolidone), Silica gel (28 to 200 mesh), and
granulated activated carbon (food grade).
The data obtained from the extraction experiments indicates that an
extraction time of 30 minutes is sufficient to remove all
extractive proteins from the meal. Beyond 30 minutes, no
significant increase in extracted protein is seen at any of the pH
or saline levels tested. The following Table I shows the amounts of
extracted protein obtained at each pH and salt level:
TABLE-US-00001 TABLE I Extracted Protein (g/L) at each pH and Salt
Level at 60 minutes 0.0 M 0.05 M 0.10 M 0.15 M 0.25 M pH 6.3 5.63
8.00 8.20 9.16 7.4 pH 8.0 4.97 6.66 9.50 9.29 8.7 pH 9.8 7.90 10.68
10.77 10.9 10.7 pH 11.0 12.0 12.56 12.91 12.36 12.93
The following Tables II to VI show the effect of salt concentration
on extracted protein (amounts in g/L) as a function of time at
various pH levels:
TABLE-US-00002 TABLE II Saline Used: 0.0 M pH 6.3 pH 8.0 pH 9.8 pH
11.0 T10 3.75 3.94 6.6 8.4 T20 4.44 4.69 6.3 9.9 T30 4.39 4.01 8.1
12.6 T40 5.11 5.67 8.2 10.7 T50 4.95 5.55 8.1 11.7 T60 5.63 4.97
7.9 12
TABLE-US-00003 TABLE III Saline Used: 0.05 M pH 6.3 pH 8.0 pH 9.8
pH 11.0 T10 7.3 5.87 8 10.5 T20 8.3 8.11 9.5 12.04 T30 7.4 6.6 9.6
12.7 T40 7.5 7.3 10 12 T50 7.9 7.3 10.7 13 T60 8 6.7 10.7 12.6
TABLE-US-00004 TABLE IV Saline Used: 0.10 M pH 6.3 pH 8.0 pH 9.8 pH
11.0 T10 9.2 9.7 8.4 14.4 T20 8.4 9.6 10.13 13.17 T30 9.2 9 11.25
12.4 T40 9.3 8.9 11.23 12.57 T50 8.9 9.5 11.83 13.18 T60 8.2 9.5
10.77 12.91
TABLE-US-00005 TABLE V Saline Used: 0.15 M pH 6.3 pH 8.0 pH 9.8 pH
11.0 T10 8.71 7.05 11.65 9.05 T20 9.47 8.42 11.46 10.28 T30 9.36
8.27 10.93 11.31 T40 9.74 9.08 10.36 11.19 T50 10.24 8.36 10.72
11.54 T60 9.16 9.29 10.7 12.36
TABLE-US-00006 TABLE VI Saline Used: 0.25 M pH 6.3 pH 8.0 pH 9.8 pH
11.0 T10 7.2 7.9 11.65 11.18 T20 7.1 7.8 11.46 11.42 T30 7.5 8.3
10.93 12.44 T40 7.4 8.7 10.36 11.87 T50 7.3 8.2 10.72 12.58 T60 7.4
8.7 10.7 12.93
As may be seen from these Tables, higher pH extractions yielded
higher protein contents at each salt level while increasing salt
content beyond 0.05 M did not increase protein solubility, in the
experiments performed.
The following Table VII shows the absorbance at 330 nm of the
protein extract at each pH and salt level:
TABLE-US-00007 TABLE VII The Effects of pH on A330 at Five
Different Saline Concentrations 0.0 M 0.05 M 0.10 M 0.15 M 0.25 M
pH 6.3 21.2 23.6 58.5 48.8 51.6 pH 8.0 27.2 42.1 66.8 52 52.5 pH
9.8 32.8 39.7 36.2 31.9 27.9 pH 11.0 43.1 42.1 42.8 36.9 42.9
As may be seen from Table VII, a reduction in extracted A330 colour
occurs at pH 9.8 in extractions of 0.1 M, 0.15 M and 0.25 M. At
this pH, the colour looks visibly darker than at other pH values
and there is no corresponding drop in protein content. As noted
above, the protein content of these extractions continues to rise
through pH 9.8 and pH 11.0.
The following Table VIII shows the absorbance at 330 nm of the
protein solution following ultrafiltration and prior to
diafiltration while Table IX shows the A330 of the protein solution
after diafiltration. As may be seen from these Tables, in each run,
the A330 of the retentate was lower after having been
diafiltered.
TABLE-US-00008 TABLE VIII Retentate A330 prior to Diafiltration 0.0
M 0.05 M 0.10 M 0.15 M 0.25 M pH 6.3 41.7 49.3 71.4 63.5 67.6 pH
8.0 59.8 52 77.4 63.4 66.2 pH 9.8 39.9 61.6 59.1 45 38.5 pH 11.0
50.9 70.4 56.4 55.4 60.1
TABLE-US-00009 TABLE IX Retentate A330 following Diafiltration 0.0
M 0.05 M 0.10 M 0.15 M 0.25 M pH 6.3 29.8 18.0 15.0 22.2 17.6 pH
8.0 37.9 22.7 36.1 22.5 22.1 pH 9.8 15.2 38.8 25.1 34.4 19.9 pH
11.0 34.8 35.1 34.4 31 40.1
The following Table X shows the percentage reduction in A330
achieved using diafiltration.
TABLE-US-00010 TABLE X % Reduction in A330 by Diafiltration 0.0 M
0.05 M 0.10 M 0.15 M 0.25 M pH 6.3 28.5 63.5 79.0 65.0 74.0 pH 8.0
36.6 56.3 53.4 64.5 66.6 pH 9.8 61.9 37 57.5 45.8 48.3 pH 11.0 31.6
50.1 39.5 44.0 33.3
As may be seen from Table X, the greatest reduction in A330 value
achieved following UF/DF came from 0.1 M extractions at pH 6.3. Of
the five different saline levels tested, the lowest A330 value for
all but one was achieved by extraction at pH 6.3.
The following Table XI shows the A330/g/L protein ratio to take
into account different protein concentrations of final retentates.
With this ratio, a low A330 and a high protein content indicated by
a low resulting member is most desirable.
TABLE-US-00011 TABLE XI A330/g/L for Retentates Following
Diafiltration 0.0 M 0.05 M 0.10 M 0.15 M 0.25 M pH 6.3 4.79 0.76
0.60 0.69 0.59 pH 8.0 4.99 0.82 1.06 0.80 0.54 pH 9.8 1.31 1.48
1.10 0.61 n.a. pH 11.0 0.69 0.80 0.96 0.83 0.67
As may be seen from Table XI, when the A330 to protein ratio is
taken into account, the best results came from the 0.25 M saline
series for each pH level tested, with the overall lowest
A330/protein ratio coming from the 0.25 M extraction at pH 8.0, in
the experiments performed.
Examination of the permeate A330 data (not shown) from both the
ultrafiltration and the diafiltration suggests that more A330 is
flushed out through the permeate at the two lower pH levels than at
pH 9.8 and 11.0.
In testing the adsorbents, at pHs of 6.3 and 8.0, Polyclar reduced
the free phenolics (A330) of the retentates and did not show a loss
in protein following the adsorption step. However, Polyclar at pH
9.8 and 11.0 did not recover significant amounts of free phenolics.
Amberlite XAD reduced A330 in most cases, run at high pH, but
protein also was lost, in about every case.
Of the other adsorbents tested, silica gel failed to reduce the
A330 in most cases and quite often made the sample cloudy, leading
to a higher A330 reading. Amberlite SF120 showed some reduction in
A330 at lower pH levels but again did not appear to be as effective
at the higher pH levels and in many cases showed a significant loss
in protein. These samples also had some precipitation after passing
through the adsorbent.
The granulated activated carbon (GAC) worked quite well at reducing
A330 in the retentates at lower pH levels but did not effectively
reduce A330 at pH 9.8 and 11.0. The GAC also exhibited some protein
loss for most of the tests. The samples that had been passed
through the GAC had to be filtered with a 0.45 .mu.M filter
following treatment owing to the presence of residual carbons.
Example 2
This Example illustrates the effect of addition of an anti-oxidant
on the extraction step.
The procedure of Example 1 was repeated in which extractions were
performed at pH 8.0 and pH 6.3 in 0.1 M saline with the addition of
ascorbic acid and with purging of extraction medium with helium to
remove 99% of the dissolved oxygen. A420 absorption was also
determined as a measure of visible colour.
The following Table XII shows the extraction data:
TABLE-US-00012 TABLE XII Extraction Data: Extracted Extracted
Extracted Protein g/L A330 A420 0.1 M, pH 8.0 11.07 38.3 11.29 0.1
M, pH 8.0, 0.01% ascorbic 11.34 47.5 5.41 0.1 M, pH 8.0, 0.05%
ascorbic 12.18 47.2 4.96
As may be seen from Table XII, the use of ascorbic acid in the
extraction reduces visible colour as shown by A420. Low levels of
ascorbic acid (0.05%) can result in greater than a two-fold
reduction in extraction A420, or visible colour.
The following Table XIII shows the diafiltration retentate A330 and
A420 readings:
TABLE-US-00013 TABLE XIII Retentate A330 and A420 Readings UF DF DF
DF Retentate Retentate Retentate Retentate Retentate A330/ A330
A330 A420 g/L Protein Protein Ratio 0.1 M, 44.1 14.8 4.3 27.6 0.54
pH 8.0 0.1 M, 58.2 16.5 3.29 30.6 0.54 pH 8.0, 0.01% ascorbic 0.1
M, 60.4 18.8 3.41 37.4 0.50 pH 8.0, 0.05% ascorbic
As may be seen from Table XIII, the reduction in A420 by ascorbic
acid in the extraction is still reflected after diafiltration. The
A420 of retentates from extractions with ascorbic acid were lower
than retentates that did not have ascorbic acid in the
extraction.
Table XIV shows the effect of Polyclar on A330 and A420 in
retentates:
TABLE-US-00014 TABLE XIV A330 A330 % A420 A420 Before After A330
Before After % A420 Treat- Treat- Reduc- Treat- Treat- Reduc- ment
ment tion ment ment tion 0.1 M, pH 8.0 14.8 11.9 19.6 4.3 3.25 24.5
0.1 M, pH 8.0, 16.5 10.1 38.8 3.29 2.41 26.8 0.01% ascorbic 0.1 M,
pH 8.0, 18.8 13.5 28.2 3.41 2.78 18.5 0.05% ascorbic
As may be seen from Table XIV, reduced A420 by ascorbic acid used
in the extraction is still present even after treatment with an
adsorbent. Polyclar reduced the A420 of each sample, but the two
samples containing ascorbic acid were lower than the control
without ascorbic acid.
Example 3
This Example also illustrates the effect of salt concentration and
pH on the extraction with an anti-oxidant.
This Example is a repeat of Example 1, except that 0.5 g (0.1%) of
sodium sulfite (Na.sub.2SO.sub.3) was added to the canola oil seed
meal extraction liquid prior to commencement of the extraction
step. All other parameters used were the same as in Example 1,
except that the diavolume value was 5.
The following Tables XV.1 to XV.5 show the amounts of protein
obtained at each pH and salt level:
TABLE-US-00015 TABLE XV.1 Extraction rate of runs with 0.0 M NaCl
and 0.1% Na.sub.2SO.sub.3 (g/L) Time (min) pH 6.3 pH 8.0 pH 9.8 pH
11.0 10 4.5 11.0 11.4 12.2 20 5.6 6.1 8.8 14.3 30 6.2 6.0 10.0 14.2
40 6.6 6.1 11.0 14.1 50 7.8 6.4 10.9 14.5 60 6.2 6.8 11.1 13.7
TABLE-US-00016 TABLE XV.2 Extraction rate of runs with 0.05 M NaCl
and 0.1% Na.sub.2SO.sub.3 (g/L) Time (min) pH 6.3 pH 8.0 pH 9.8 pH
11.0 10 9.2 8.9 8.9 11.1 20 8.7 8.7 9.9 11.1 30 9.2 8.5 9.0 12.2 40
9.1 8.4 10.6 12.4 50 9.5 8.5 10.0 13.0 60 10.5 7.3 10.9 13.8
TABLE-US-00017 TABLE XV.3 Extraction rate of runs with 0.10 M NaCl
and 0.1% Na.sub.2SO.sub.3 (g/L) Time (min) pH 6.3 pH 8.0 pH 9.8 pH
11.0 10 8.4 8.3 9.2 11.3 20 9.7 9.1 10.3 11.5 30 10.1 9.0 10.3 11.3
40 9.2 9.0 9.8 11.4 50 10.4 9.4 10.0 12.3 60 10.1 9.1 10.8 11.3
TABLE-US-00018 TABLE XV.4 Extraction rate of runs with 0.15 M NaCl
and 0.1% Na.sub.2SO.sub.3 (g/L) Time (min) pH 6.3 pH 8.0 pH 9.8 pH
11.0 10 8.8 11.0 11.2 13.0 20 9.5 10.6 12.5 13.7 30 8.3 11.4 12.6
14.0 40 8.8 10.8 12.4 14.4 50 9.6 10.5 12.7 13.6 60 9.8 10.6 12.5
14.1
TABLE-US-00019 TABLE XV.5 Extraction rate of runs with 0.25 M NaCl
and 0.1% Na.sub.2SO.sub.3 (g/L) Time (min) pH 6.3 pH 8.0 pH 9.8 pH
11.0 10 11.5 10.7 12.7 12.3 20 11.0 12.8 14.0 13.0 30 12.1 13.4
14.8 13.4 40 11.8 18.4 14.6 13.1 50 12.3 12.4 14.7 14.1 60 12.2
13.4 15.2 14.4
As may be seen from these Tables, extraction reached equilibrium in
about 30 minutes in most runs. When no salt was added, more protein
was extracted as the pH was raised. The effect of pH seemed less
significant at pH below 8.0 than at high pH above 9.8 (Table XV.1).
Salt addition at low levels (less than 0.10 M) was able to
substantially increase the protein extractability at pH 6.3 and
8.0, but low salt concentrates did not assist protein extraction at
higher pH levels of 9.8 or 11.0 (Tables XV.2 and XV.3).
The following Tables XVI shows the effect of pH and sodium chloride
concentration on the free phenolic content (A330 absorbance) of the
protein extract:
TABLE-US-00020 TABLE XVI Effect of pH and NaCl concentration on
A330 of protein extract (with Na.sub.2SO.sub.3) 0.25 M 0 NaCl 0.05
M NaCl 0.10 M NaCl 0.15 M NaCl NaCl pH 6.3 41.1 49.1 34.5 51.1 54.2
pH 8.0 24.3 36 38.3 39.9 41.8 pH 9.8 30 28.2 27.5 27.8 29 pH 11.0
38.4 29.7 32.7 31.5 32.1
As seen in this Table XVII, the A330 showed a decreasing value with
rising pH to pH 9.8 although the protein concentration also
increased over this range. The colour extract became visibly darker
as the pH rose. The salt concentration had a less pronounced effect
on colour.
The following Tables XVII.1 to XVII.4 show the effect of pH and
NaCl concentration on A330 of retentate (Table XVII.1) and permeate
(Table XVII.2) from ultrafiltration and on A330 of retentate (Table
XVII.3) and permeate (Table XVII.4) from diafiltration.
TABLE-US-00021 TABLE XVII.1 Effect of pH and NaCl concentration on
A330 of retentate from ultrafiltration (with Na.sub.2SO.sub.3) 0.25
M 0 NaCl 0.05 M NaCl 0.10 M NaCl 0.15 M NaCl NaCl pH 6.3 88.7 71.9
44.4 73.2 75.3 pH 8.0 18.3 55.2 60.0 62.3 65.5 pH 9.8 55.9 48.1
59.1 49.7 54.2 pH 11.0 77.6 57.4 56.4 61.8 62.3
TABLE-US-00022 TABLE XVII.2 Effect of pH and NaCl concentration on
A330 of permeate from ultrafiltration (with Na.sub.2SO.sub.3) 0
NaCl 0.05 M NaCl 0.10 M NaCl 0.15 M NaCl 0.25 M NaCl pH 6.3 31.3
40.6 24.8 43.4 41.8 pH 8.0 16.0 35.9 29.8 34.4 30.1 pH 9.8 25.2
20.6 23.5 23.9 24.0 pH 11.0 25.1 21.3 23.3 25.4 25.1
TABLE-US-00023 TABLE XVII.3 Effect of pH and NaCl concentration on
A330 of retentate from diafiltration (with Na.sub.2SO.sub.3) 0 NaCl
0.05 M NaCl 0.10 M NaCl 0.15 M NaCl 0.25 M NaCl pH 6.3 34.8 24.7
14.9 21.2 21.2 pH 8.0 13.9 17.8 20.8 20.0 23.7 pH 9.8 30.6 34.3
32.3 20.2 22.1 pH 11.0 58.5 29.0 24.8 35.0 24.5
TABLE-US-00024 TABLE XVII.4 Effect of pH and NaCl concentration on
A330 of permeate from diafiltration (with Na.sub.2SO.sub.3) 0 NaCl
0.05 M NaCl 0.10 M NaCl 0.15 M NaCl 0.25 M NaCl pH 6.3 7.0 8.5 6.3
7.9 8.5 pH 8.0 3.1 10.0 6.0 6.5 5.7 pH 9.8 8.3 8.0 6.9 7.0 5.7 pH
11.0 6.8 5.4 5.1 5.8 5.2
Since ultrafiltration concentrated the protein in the extract four
times, the retentate was visibly much darker and had a higher A330
reading than the extract (Table XVII.1) except for 0.0 NaCl at pH
8.0, but the latter may be an anomalous result. Similar to the
original extract before UF, a minimum in A330 occurred at pH 9.8,
which was not supported by the actual visible colour darkness for
reasons previously discussed. Measured at A330, UF recovered a
substantial amount of the phenolics from the extract as shown by
the high A330 reading in the permeate (see Table XVII.2).
From Table XVII.3, it can be seen that diafiltration retentate had
a much lower A330 reading than the UF retentate (Table XVII.1).
Although the DF permeate (Table XVII.4) was not as high in A330
reading as that of UF permeate (Table XVII.4), the DF nevertheless
resulted in the further removal of considerable amounts of the
remaining phenolics. This additional removal of phenolics by
diafiltration resulted in a much lower A330 in the DF retentate
(Table XVII.3) than in the UF retentate (Table VII.1).
The following Table XVIII.1 to XVIII.4 show the effect of
adsorbents and pH on A330 of retentate:
TABLE-US-00025 TABLE XVIII.1 Effect of adsorbents and pH on A330 of
retentate (0.05 M NaCl with 0.1% Na.sub.2SO.sub.3) pH Control
Polyclar XAD SF 120 Silica gel 6.3 24.7 14.8 21.7 19.5 20.8 8.0
17.8 13.8 15.8 18.8 19.9 9.8 34.3 30.8 32.4 38.6 41.2 11.0 29 28.7
25.1 29.8 30.5
TABLE-US-00026 TABLE XVIII.2 Effect of adsorbents and pH on A330 of
retentate (0.10 M NaCl with 0.1% Na.sub.2SO.sub.3) pH Control
Polyclar XAD SF 120 Silica gel GAC 6.3 14.9 10.9 9.5 14.6 12.6 11.8
8.0 20.8 15.8 16.6 19.9 22.7 19.1 9.8 32.3 22.7 32 31.1 36.2 26.4
11.0 24.8 23.3 23.2 26.5 28.2 26.6
TABLE-US-00027 TABLE XVIII.3 Effect of adsorbents and pH on A330 of
retentate (0.15 M NaCl with 0.1% Na.sub.2SO.sub.3) pH Control
Polyclar XAD SF 120 Silica gel GAC 6.3 21.2 13.5 15.7 18.3 18.7
19.4 8.0 20 16 16.3 17.9 19.5 19.5 9.8 20.2 18 16.1 21 29.8 20 11.0
35 28 31.6 33.6 35.2 34.2
TABLE-US-00028 TABLE XVIII.4 Effect of adsorbents and pH on A330 of
retentate (0.25 M NaCl with 0.1% Na.sub.2SO.sub.3) pH Control
Polyclar XAD SF 120 Silica gel GAC 6.3 21.2 15.9 16.6 21.4 20.4
21.4 8.0 23.7 16.1 21 24.5 25.7 24.5 9.8 22.1 19.1 21.1 22.9 26.5
22.7 11.0 24.5 22.8 18.4 24.2 27.5 25.1
As may be seen from Tables XVIII.1 to XVIII.4, at low pH (<9.8),
Polyclar, among all adsorbents tested, was particularly effective
in decreasing the A330 reading in the final retentate. As seen in
Table XVIII.1, the A330 value may be reduced by up to 40%.
Although other adsorbents were also able to lower A330 readings
under specific conditions of pH and salt concentration, their
effect was somewhat insignificant when compared to that of
Polyclar. When pH of 9.8 was used, Polyclar was less useful in
lowering A330.
The following Tables XVIII.5 to XVIII.8 show the effect of
absorbents on protein concentration (g/L) in the retentate:
TABLE-US-00029 TABLE XVIII.5 Effect of adsorbents and pH on protein
concentration of retentate (0.05 M with 0.1% Na.sub.2SO.sub.3) pH
Control Polyclar XAD SF 120 Silica gel 6.3 51.5 53.2 47.1 46.3 46.5
8.0 39 46 37.2 48.5 41.4 9.8 40.4 41.1 38.5 38.3 41.1 11.0 48.2
51.5 47.8 50.6 49.1
TABLE-US-00030 TABLE XVIII.6 Effect of adsorbents and pH on protein
concentration of retentate (0.10 M with 0.1% Na.sub.2SO.sub.3) pH
Control Polyclar XAD SF 120 Silica gel GAC 6.3 43.6 46 41.4 43.9
45.7 44.3 8.0 50.5 55.1 51.7 50.3 52 52.8 9.8 42.5 47.3 43.9 44.2
45.4 46 11.0 38 42 36.3 38.2 39.4 39.8
TABLE-US-00031 TABLE XVIII.7 Effect of adsorbents and pH on protein
concentration of retentate (0.15 M with 0.1% Na.sub.2SO.sub.3) pH
Control Polyclar XAD SF 120 Silica gel GAC 6.3 33.5 36.2 32.3 34.7
36.9 33.3 8.0 46.8 48.7 44.2 46.4 48.2 47.4 9.8 39.5 40.9 36.2 39.7
39.7 40.4 11.0 56.2 59.5 50.4 55.8 60.1 58.5
TABLE-US-00032 TABLE XVIII.8 Effect of adsorbents and pH on protein
concentration of retentate (0.25 M with 0.1% Na.sub.2SO.sub.3) pH
Control Polyclar XAD SF 120 Silica gel GAC 6.3 38.8 41.7 36.2 38.4
40.7 39 8.0 44.7 46.4 41.4 43.5 45.8 45.8 9.8 50.3 54 48.8 50.5
52.2 51.7 11.0 41.9 47.2 38.6 41.4 44.6 42.5
As may be seen from Tables XIVIII.5 to XVIII.8, all adsorbents
tested were quite inert to protein concentration in the retentate
at all combinations of salt and pH, even though the protein was
much concentrated by ultrafiltration.
Example 4
This Example describes the effect of using lower levels of sodium
sulfite and of purged extraction.
The procedure of Example 3 was repeated employing a lower level of
sodium sulfite (0.05 wt % Na.sub.2SO.sub.3) for three runs at pH
8.0 using Polyclar as the adsorbent. A420 was measured in addition
to A330.
In another set of experiments, the extract solution containing 0.05
wt % Na.sub.2SO.sub.3 also was purged with helium before and during
extraction.
The following Table XIX.1 shows the effect of these modifications
on protein extraction:
TABLE-US-00033 TABLE XIX.1 Protein extraction at pH 8.0 (g/L) 0.1%
Na.sub.2SO.sub.3 0.05% Na.sub.2SO.sub.3 He purge 0.05 M NaCl 8.5
11.8 11.5 0.10 M NaCl 9.0 12.7 12.0 0.15 M NaCl 11.4 14.3 14.5
As may be seen from Table XIX.1, at all three salt addition levels,
protein concentration in the extract increased by about 40 wt %
when reduced quantities of sodium sulfite were used. The higher
salt concentration led to a higher protein concentration. The
helium purge had no bearing on protein extraction.
Table XIX.2 shows the effect of these modifications on colour at
A330:
TABLE-US-00034 TABLE XIX.2 Absorbance of extract at 330 nm 0.1%
Na.sub.2SO.sub.3 0.05% Na.sub.2SO.sub.3 He purge 0.05 M NaCl 36.0
40.5 35.9 0.10 M NaCl 38.3 36.6 42.3 0.15 M NaCl 39.9 43.4 42.7
As may be seen from Table XIX.2, the reduction in Na.sub.2SO.sub.3
did not significantly affect the A330 of the extract. Although the
helium purge removed 99% of the dissolved oxygen, the absorbance of
the extract was not improved either at 330 nm or 420 nm (Table
XIX.3 below):
TABLE-US-00035 TABLE XIX.3 Absorbance of extract at 420 nm No purge
He purge 0.05 M NaCl 9.0 9.4 0.10 M NaCl 8.8 7.1 0.15 M NaCl 8.4
10.0
The following Table XX shows the effect of membrane processing on
the A330 and A420 of retentate:
TABLE-US-00036 TABLE XX Effect of membrane processing on the A330
and A420 of retentate Extract UF retentate DF retentate Salt A330
A420 A330 A420 A330 A420 0.05 M 40.5 9.0 57.9 10.9 20.7 4.1 0.10 M
36.6 8.8 55.9 10.9 15.1 3.5 0.15 M 43.4 8.4 69.9 11.4 20.2 5.4 0.05
M w He* 35.9 9.4 55.3 12.4 18.3 4.0 0.10 M w He 42.3 7.1 67.0 10.4
20.5 4.7 0.15 M w He 42.7 10.0 61.3 12.9 20.2 4.3 *with helium
purge
As seen in Table XX, diafiltration substantially removed the
coloured components in the extract. Both A330 and A420 readings for
the final retentate were about half those of the original extract.
Helium purge had no effect on A330 or A420 values.
The following Table XXI shows the effect of Polyclar on A330 and
A420 of retentate:
TABLE-US-00037 TABLE XXI Effect of Polyclar on A330 and A420
Control Polyclar* Salt A330* A420* A330 A420 0.05 M 20.7 4.1 14.9
(28%) 3.6 (12%) 0.10 M 15.1 3.5 11.3 (25%) 2.7 (23%) 0.15 M 20.2
5.4 16.1 (20%) 4.6 (15%) 0.05 M w He** 18.3 4.0 12.5 (32%) 3.6
(10%) 0.10 M w He 20.5 4.7 18.5 (10%) 4.1 (13%) 0.15 M w He 20.2
4.3 13.9 (31%) 3.8 (12%) *Numbers in brackets are the percentages
of reduction **with helium purge
Example 5
This Example illustrates the preparation of a canola protein
isolate from commercial canola meal using an antioxidant and
diafiltration.
150 kg of commercial canola meal (higher temperature toasted meal)
was added to 1000 L of 0.15 M NaCl containing 0.5 kg (0.05 wt %)
ascorbic acid solution at 16.degree. C. and agitated for 30 minutes
to provide an aqueous protein solution having a protein content of
20.2 g/L. The residual canola meal was removed and washed on a
vacuum filter belt. The resulting protein solution was clarified by
centrifugation and filtration to produce 1040 L of a clarified
protein solution having a protein content of 14.6 g/L.
The protein extract solution was reduced in volume to 45 L by
concentration on an ultrafiltration system using 5000 dalton
molecular weight cut-off membranes. The protein extract solution
then was diafiltered on a diafiltration system using 5000 dalton
molecular weight cut-off membranes with 450 L of 0.15 M NaCl
solution containing 0.05 wt % ascorbic acid to a final volume of 44
L with a protein content of 225 g/L.
The concentrated and diafiltered solution at 30.degree. C. was
diluted 1:15 into 4.degree. C. water. A white cloud of protein
micelles formed immediately and was allowed to settle. The upper
diluting water was removed and the precipitated, viscous, sticky
mass (PMM) was recovered from the bottom of the vessel and dried.
The dried protein was found to have a protein content of 103.2 wt %
(N.times.6.25) d.b.
620 L of supernatant from the micelle formation were concentrated
to 30 L by concentration on an ultrafiltration system using 5000
dalton molecular weight cut-off membranes. The concentrated
supernatant then was diafiltered on a diafiltration system using
5000 dalton molecular weight cut-off membranes with 100 L of water
to a final volume of 27 L with a protein content of 121.8 g/L.
The concentrated and diafiltered solution was dried. The dried
protein was found to have a protein content of 100.8 wt %
(N.times.6.25) d.b.
Samples of the PMM-derived canola protein isolate (CPI) and the
supernatant-derived canola protein isolate were evaluated for
lightness (L) and chromaticity (a and b) using a Minolta (CR-310)
colorimeter. In the Lab space, the value moves from 0 to 100, with
100 being white and 0 being black. The chromaticity coordinates, a
and b, both have maximum values of +60 and -60, +a being the red
direction, -a being the green direction, +b being the yellow
direction and -b being the blue direction.
The following Table XXII shows the results obtained:
TABLE-US-00038 TABLE XXII Sample L a b PMM-derived CPI 83.08 -1.58
+27.89 Supernatant-derived CPI 79.38 -0.11 +20.46
The canola protein isolates exhibited a lighter (L) and less yellow
(b) colour than isolates produced following this procedure but
omitting the addition of ascorbic acid (as an antioxidant) and the
diafiltration steps (data not shown).
Example 6
This Example illustrates the effect of temperature on the colour of
protein extracts from a low temperature toasted meal and an
air-desolventized meal.
75 g samples of a (a) low-temperature toasted (100.degree. C.)
canola oil seed meal (LT) and (b) an air-desolventized (20.degree.
C.) canola oil seed meal (Marc) were added to 500 samples of 0.15 M
NaCl solution at ambient or room temperature (RT), 55.degree. C.,
60.degree. C. and 65.degree. C., agitated for 30 minutes while
maintaining the temperature of the solution substantially constant
to provide aqueous protein solutions. Samples of the aqueous
protein solution were taken at 5, 10, 15, 20 and 30 minutes for
analysis. The spent meal was separated by centrifugation at
10,000.times.g for 5 minutes and freeze dried.
Absorbances at A330 and A420 were determined for the various
protein solution samples. As already noted above UV absorbance at
A330 is indicative of phenolics concentration in solution while
absorbance at A420 is more direct measurement of actual colour. The
data for the various samples are set forth in the following Tables
XXIII AND XXIV:
TABLE-US-00039 TABLE XXIII Absorbance Readings for Extracts of
Low-temperature Meal Extraction RT 55.degree. C. 60.degree. C.
65.degree. C. Time (min) A330 A420 A330 A420 A330 A420 A330 A420 5
80.7 1.96 97.2 2.70 99.8 3.03 98.3 2.84 10 88.9 2.42 92.0 2.77 98.0
3.07 93.9 2.95 15 92.5 2.50 89.1 2.94 95.6 3.10 93.0 3.05 20 90.7
2.55 86.1 2.90 93.7 3.23 90.7 3.16 30 90.7 2.56 88.2 3.22 97.8 3.47
88.1 3.24
TABLE-US-00040 TABLE XXIV Absorbance Readings for Extracts of Marc
Meal Extraction RT 55.degree. C. 60.degree. C. 65.degree. C. Time
(min) A330 A420 A330 A420 A330 A420 A330 A420 5 120.3 2.73 118.8
2.94 120.5 3.08 128.4 3.16 10 116.7 2.73 118.5 3.07 121.1 3.15
127.4 3.19 15 117.0 2.78 119.5 3.20 116.3 3.09 112.5 3.04 20 119.1
2.84 113.1 3.17 112.9 3.19 121.6 3.09 30 114.1 2.74 113.1 3.23
114.6 3.22 119.1 3.00
As may be seen from Tables XXIII and XXIV, elevating the extraction
temperature had no significant effect on the A330 of the extracted
protein solution for each meal type tested but there was a slight
increase in the A420 readings seen at higher temperatures.
Example 7
This Example shows the effects of certain parameters on colour of
protein extracts from certain canola oil seed meals.
In a first set of experiments, 50 g samples of canola oil seed meal
which (a) had been low temperature toasted at 100.degree. C. (LT
meal) or (b) which had been air-desolventized at 20.degree. C.
(Marc meal) were added to 500 mL samples of 0.05 M or 0.10 M NaCl
solution at room temperature (20.degree. C.) and stirred for 15
minutes. The slurry was centrifuged at 5000.times.g for 10 minutes
to remove spent meal.
In a second set of experiments, 500 mL of water with no salt added
was first heated to 60.degree. C. on a hot plate stirrer. Then 50 g
of canola oil seed meal, which had been low temperature toasted at
100.degree. C., or (b) which had been air-desolventized at
20.degree. C. (Marc meal), was added and stirred for 15 minutes
while the temperature was maintained. The extract was separated
from the spent meal by centrifugation at 5000.times.g for 10
minutes.
Absorbances at A330 and A420 and protein concentrations were
determined for the various protein solutions. The results obtained
are set forth in the following Table XXV.1 and XXV.2:
TABLE-US-00041 TABLE XXV.1 Absorbance Readings for Extracts 0.05 M
saline 0.10 M saline 60.degree. C. water A330 A420 A330 A420 A330
A420 LT meal 62.4 1.88 64.4 1.84 55.4 2.10 Marc meal 77.7 1.82 85.5
2.10 78.0 2.13 0.05 M Saline 0.1 M Saline 60.degree. C. Water LT
Meal 1.11 1.44 0.98 Marc Meal 2.09 2.04 1.38
As may be seen from the results contained in Table XXV.1 and XXV.2,
the A330 values increase with increasing protein concentration
while colour intensity, as indicated by A420, did not change
significantly with the protein concentration, coinciding with
visual observation. These results show that, along with a higher
protein yield, a lighter product may be expected from the
air-desolventized meal in comparison with low temperature toasted
meal.
Example 8
This Example shows the effect of solvent extraction of canola
protein isolate on product colour.
A mixture of PMM-derived canola protein isolates was formed from
three isolation procedures, such PMM-derived isolates being D29-02A
C300 (57.9 wt %), D24-02A C300 (34.7 wt %) and D11-02A C300 (7.4 wt
%) (Composite 6). In addition a mixture of supernatant-derived
isolates was formed from three isolation procedures, such
supernatant-derived isolates being E29-02A C200 (18.7 wt %),
D29-02A C200 (40.1 wt %) and E14-02A C200 (41.2 wt %) (Composite
7).
The specific procedures utilized to prepare the individual canola
protein isolates are as follows:
`a` kg of commercial canola meal was added to `b` L of 0.15 M NaCl
solution at ambient temperature, agitated for 30 minutes to provide
an aqueous protein solution having a protein content of `c` g/L.
The residual canola meal was removed and washed on a vacuum filter
belt. The resulting protein solution was clarified by
centrifugation and filtration to produce `d` L of a clarified
protein solution having a protein content of `e` g/L.
A `f` L aliquot of the protein extract solution was reduced in
volume to `g` L by concentration on an ultrafiltration system using
`h` dalton molecular weight cutoff membranes. The resulting
concentrated protein solution had a protein content of `i` g/L.
The concentrated solution at `j` .degree. C. was diluted `k` into
4.degree. C. water. A white cloud formed immediately and was
allowed to settle. The upper diluting water was removed and the
precipitated, viscous, sticky mass (PMM) was recovered from the
bottom of the vessel in a yield of `l` wt % of the extracted
protein. The dried PMM derived protein was found to have a protein
content of `m` % (N.times.6.25) d.b. The product was given
designation `n`.
The following Table XXVI gives the values of the parameters `a` to
`m`:
TABLE-US-00042 TABLE XXVI BW- AL017- BW-AL017- BW-AL017- BW-AL017-
BW-AL018- D11-02A D24-02A D29-02A E14-02A E29-02A n C300 C300 C300
C300 C300 a 1200 150 150 150 150 b 8000 1000 1000 999 1001 c 26.3
25.7 20.2 20 24.4 d 5882 1152 1040 1245 1075 e 17.7 16.6 14.6 10.2
17.8 f 5882 1080 1040 1194 820 g 92 53 44.25 39 24 h 5000 5000 5000
5000 5000 i 289 246.8 225 238 289 j 31 32 32 32 32 k 1:15 1:15 1:15
1:15 1:15 l 8.5 37.3 29.4 27.7 23.9 m 104.4 105.1 103.2 100.1
102.4
The removed diluting water was reduced in volume by ultrafiltration
using a `o` dalton molecular weight cut-off membrane to a protein
concentration of `p` g/L. The concentrate was dried. With the
additional protein recovered from the supernatant, the overall
protein recovery was `q` wt % of the extracted protein. The dried
protein formed had a protein content of `r` wt % (N.times.6.25)
d.b.
The product was given designation `s`. The following Table XXVII
gives the values for the parameters `o` to `r`:
TABLE-US-00043 TABLE XXVII BW- AL017- BW-AL017- BW-AL017- BW-AL017-
BW-AL018- D11-02A D24-02A D29-02A E14-02A E29-02A s C200 C200 C200
C200 C200 o 3000 5000 5000 5000 5000 p 237.6 194.4 121.8 115.8
100.1 q 15.5 55.4 45.7 44.4 35.2 r 98.7 97.8 100.8 98.7 97.5
1.4 kg of Composite 6 was dispersed in a blend of 3L of ethanol
(denatured: 85% ethanol/15% wood alcohol, VWR Canlab D and 3L of
reverse osmosis (RO) purified water. The mixture was stirred for 30
minutes using an overhead stirrer. Solid material was separated
from the bulk liquid by centrifuging the sample in batches at 8000
g for 5 minutes.
The pellets were then redispersed and extracted again in a further
blend of 3L of ethanol and 3L of RO water for 30 minutes with
stirring. Centrifugation (8000 g/5 min.) again was used to collect
the solid sample. The pellets were then dispersed into 4L of
ethanol in an effort to remove water from the samples. The solid
material was collected by centrifugation (8000 g/5 min.) and
re-dispersed in 4L of fresh ethanol.
Centrifugation (8000 g/5 min.) again was used to collect the
solids. The pellets were broken up and spread on a baking sheet and
left in a fumehood to dry.
This procedure was repeated using 1.4 kg of Composite 7, which was
dispersed in a blend of 4.2 L of ethanol and 1.8 L of reverse
osmosis purified water. The pellets were redispersed in a fresh
blend of 4.2L of ethanol and 1.8L of RO water.
The protein powders obtained and solvent extract samples were
analyzed for total protein content and by HPLC. Protein powders
were also analyzed for moisture content. Solvent extract samples
were also examined using a spectrophotometer to give an indication
of their phenolic content (absorbance at 330 nm) and visible colour
(absorbance at 420 nm). The colour of the dry protein products was
assessed using a Minolta CR-310 colour meter.
The recovery of ethanol/water-extracted Composite 6 was 86 wt % and
for Composite 7 was 80 wt %. Product losses were due to solubility
in the extraction solvent and the following Table XXVIII gives the
protein content of the solvent extracts.
TABLE-US-00044 TABLE XXVIII Protein content of solvent extracts
Sample wt % protein Composite 6 - first extraction 1.12 Composite 6
- second extraction 0.46 Composite 7 - first extraction 1.55
Composite 7 - second extraction 1.27
Other losses are attributable to material lost due to handling of
the samples.
The colour readings obtained are set forth in the following Table
XXIX:
TABLE-US-00045 TABLE XXIX Colour readings for composite sample
before and after extraction Sample L a B Composite 6 before
extraction 81.49 +0.12 +24.37 Composite 6 after extraction 83.53
-0.56 +14.18 Composite 7 before extraction 79.68 +0.20 +19.69
Composite 7 after extraction 80.67 +0.13 +14.72
As may be seen from the data in Table XXIX, for both Composite 6
and Composite 7, the extraction of the canola protein isolate
resulted in an increase in lightness (L), a decrease in "a" value
and a decrease in "b" value. The increase in L value means the
product is more white and less black. The decrease in "a" value
corresponds to a shift in colour from red towards green while the
decrease in "b" value corresponds to a shift in colour from yellow
towards blue. The reduction in redness and yellowness of the
samples is an indication of the removal of phenolic compounds
and/or their reaction products.
In the following Table XXX provides the absorbance readings for
solvent extracts:
TABLE-US-00046 TABLE XXX Absorbance readings for solvent extracts
Sample A420 A330 Composite 6 - first extract 3.19 21.60 Composite 6
- second extract 0.82 6.00 Composite 7 - first extract 3.20 11.40
Composite 7 - second extract 0.80 3.00
As may be seen from Table XXX, the extracts are lightly coloured,
indicating extraction of colourants from the protein isolate.
Table XXXI shows the protein content (N.times.6.25. Percentage
nitrogen values were determined using a Leco FP52D Nitrogen
Determinator) and moisture content of the solvent extracted protein
isolates:
TABLE-US-00047 TABLE XXXI Characteristics of solvent extracted
protein isolates Sample Protein content (wt % w.b.) Moisture
content (wt %) Composite 6 97.35 6.13 Composite 7 94.09 3.75
As may be seen from Table XXXI, the solvent extracted products were
low in moisture and had a protein content sufficiently high for the
products to be classified as isolates
Example 9
This Example illustrates the use of an antioxidant and adsorbent in
the production of a canola protein isolate.
150 kg of a commercial canola oil seed meal which had been
desolventized at low temperature (100.degree. C.) was added to
1000L of 0.15 M NaCl and mixed for 30 minutes at a room temperature
of 21.degree. C. After 15 minutes of mixing 0.05 wt % (500 g) of
ascorbic acid was added to the slurry as an antioxidant.
The residual canola meal was removed and washed on a vacuum filter
belt resulting in 953.5 L of protein solution having a protein
content of 23.9 g/L. The UV absorbance of the solution at 330 nm
was 61.2.
21.2 kg (2.2 wt %) Polyclar Super R was added to the 953.5 L of
protein solution and allowed to mix for 1 hour at room temperature.
Thereafter, the Polyclar was removed by passing the protein
solution through a desludger centrifuge and then filter presses
containing 20 and 0.2 .mu.M filter pads, respectively. Following
Polyclar removal, 842 L of canola protein solution was collected
having a protein content of 19.9 g/L and a A330 absorbance of 33.2.
A significant drop in the A330 absorbance, therefore, was obtained,
with a very low protein loss.
The clarified solution then was concentrated to a volume of 30 L
having a protein content of 338.4 g/L and an A330 of 20.4 on an
ultrafiltration system using 5000 dalton molecular weight cut-off
membranes. The concentrated protein extract solution was
diafiltered on a diafiltration system using 5000 dalton molecular
weight cut-off membranes with 300 L of 0.15M NaCl containing 0.05
wt % ascorbic acid. The resulting 29.0 L of concentrated and
diafiltered canola protein solution had a protein content of 299.7
g/L and an A330 of 25.6.
In contrast to the results seen in Examples 1 to 3 and 5, the
diafiltration had little effect on A330, likely because the
Polyclar had already removed much of the free phenolic, from the
canola protein solution prior to the concentration step.
The concentrated and diafiltered solution at 31.degree. C. was
diluted into 15 volumes of water having a temperature of
6.4.degree. C. A white cloud of protein micelles formed immediately
and was allowed to settle for two hours. The upper diluting water
was removed and the precipitated, viscous, sticky mass (PMM) (40.6
kg) was recovered from the bottom of the vessel and spray dried.
The dried protein isolate had a protein content of 98.8 wt %
(N.times.6.25) d.b.
440 L of supernatant from the micelle formation and having a
protein content of 13.3 g/L were concentrated to 30 L by
concentration on an ultrafiltration system using 5,000 dalton
molecular weight cut-off membranes. The concentrated supernatant
then was diafiltered on a diafiltration system using 5,000 dalton
molecular weight cut-off membranes with five volumes of water. The
resulting solution had a protein content of 161.0 g/L and an A330
of 10.8.
The concentrated and diafiltered solution was dried and the dried
protein was found to have a protein content of 95.6 wt %
(N.times.6.25) d.b.
Samples of the PMM-derived canola protein isolate (CPI) and the
supernatant-derived canola protein isolate were analyzed for
lightness (L) and chromaticity (a and b) using a Minolta CR-310
calorimeter.
The following Table XXXII shows the results obtained:
TABLE-US-00048 TABLE XXXII Sample L a b PMM-derived CPI 81.64 -1.46
29.57 Supernatant-derived CPI 81.24 -0.76 21.15
Example 10
This Example illustrates the use of an antioxidant and adsorbent in
the extraction step.
Bench scale experiments were carried out in which samples of
commercial canola oil seed meal which had been desolventized at
100.degree. C. were extracted with 0.15 M NaCl for 30 minutes at a
concentration of 15 wt %. Extractions were effected with and
without the addition of Polyclar Super R at varying levels, namely
0.5 wt %, 1.0 wt %, 1.5 wt %, 2.0 wt %, 2.5 wt %, 3.0 wt %, 4.0 wt
% and 5.0 wt % and with and without the addition of 0.5 wt %
ascorbic acid. Following the extraction, the solutions were
centrifuged and then analyzed for phenolics content (A330
absorbance), visible colour (A420 absorbance) and protein
content.
The results obtained with and without ascorbic acid are set forth
in Table XXXIII and XXXIV respectively:
TABLE-US-00049 TABLE XXXIII % w/v Polyclar A330 A420 Protein g/L
Control 96.2 2.8 24.6 1.0% Polyclar 93.0 3.3 25.8 1.5% Polyclar
71.7 2.65 26.1 2.0% Polyclar 63.0 2.01 23.5 2.5% Polyclar 72.4 2.54
26.4 3.0% Polyclar 64.7 2.63 26.4 4.0% Polyclar 63.0 2.41 28.0 5.0%
Polyclar 61.6 2.39 25.9
TABLE-US-00050 TABLE XXXIV % w/v Polyclar A330 A420 Protein g/L
Control 90.5 3.12 25.9 1.0% Polyclar 82.7 2.22 28.4 1.5% Polyclar
90.6 2.59 32.8 2.0% Polyclar 83.5 2.32 26.8 2.5% Polyclar 80.2 2.09
27.1 3.0% Polyclar 68.1 2.25 26.5 4.0% Polyclar 66.5 2.62 29.9 5.0%
Polyclar 52.4 1.73 26.0
As may be seen from the results set forth in the Tables XXXIII and
XXXIV, significant reduction in both A330 and A420 were obtained in
the presence of Polyclar both with and without the presence of
ascorbic acid. A 2.5% w/v concentration of Polyclar achieved a 25%
reduction in the A330 and an 11% reduction in the A420 seen in the
control, when no ascorbic acid was added to the extraction. Higher
levels of Polyclar further reduced the A330 and A420 values. With
ascorbic acid present during the extraction, a 12% reduction in
A330 and 34% reduction in A420 were seen when 2.5% w/v of Polyclar
was used. The protein content of the solution was unaffected by the
presence or absence of the Polyclar.
Example 11
This Example illustrates the effect of ethanol washing of canola
oil seed meal on colour.
10 g samples of dehulled canola oil seed meal were mixed with 100
ml of ethanol and allowed to mix for 30 minutes at 45.degree. C.,
40.degree. C. and room temperature, which was regulated with a
circulating water bath and a jacketed vessel.
After the 30 minutes mixing period, the meal/solvent slurry was
poured through a filter to separate the meal from the extracts. The
procedure was repeated until no more colour was removed or until
the A330 and A420 absorbance readings began to plateau. The meal
from each solvent wash/extraction step was dried and 0.15 M NaCl
protein extractions at ambient temperature for 30 minutes were
performed.
UV absorbance of the extract solution was performed for each
extract sample. Tables XXXV, XXXVI and XXXVII show the A330 and
A420 values for the extraction done at room temperature, 40.degree.
C. and 45.degree. C., respectively:
TABLE-US-00051 TABLE XXXV Room temperature Ethanol Extraction A330
A420 1.sup.st Solvent Extraction 34.3 0.494 2.sup.nd Solvent
Extraction 12.8 0.232 3.sup.rd Solvent Extraction 6.1 0.100
4.sup.th Solvent Extraction 2.8 0.047 5.sup.th Solvent Extraction
2.3 0.045 6.sup.th Solvent Extraction 1.7 0.040
TABLE-US-00052 TABLE XXXVI 40.degree. C. Ethanol Extraction A330
A420 1.sup.st Solvent Extraction 30.9 0.528 2.sup.nd Solvent
Extraction 20.1 0.257 3.sup.rd Solvent Extraction 9.3 0.139
4.sup.th Solvent Extraction 7.01 0.108 5.sup.th Solvent Extraction
4.74 0.073 6.sup.th Solvent Extraction 6.66 0.063 7.sup.th Solvent
Extraction 3.69 0.035 8.sup.th Solvent Extraction 2.57 0.031
9.sup.th Solvent Extraction 2.6 0.037 10.sup.th Solvent Extraction
2.4 0.033
TABLE-US-00053 TABLE XXXVII 45.degree. C. Ethanol Extraction A330
A420 1.sup.st Solvent Extraction 43.0 0.610 2.sup.nd Solvent
Extraction 21.0 0.301 3.sup.rd Solvent Extraction 14.6 0.191
4.sup.th Solvent Extraction 7.83 0.129 5.sup.th Solvent Extraction
7.65 0.105 6.sup.th Solvent Extraction 7.34 0.092 7.sup.th Solvent
Extraction 5.76 0.086 8.sup.th Solvent Extraction 6.04 0.063
9.sup.th Solvent Extraction 4.89 0.065 10.sup.th Solvent Extraction
4.98 0.052
As may be seen from this data, solvent extraction at lower
temperature did not remove as many colour and phenolic compounds as
temperatures in the 40.degree. to 45.degree. C. range, with the
room temperature extraction ceasing to remove contaminants after
only 6 extractions while the higher temperature extraction each
removed contaminants until the 10.sup.th extraction.
The protein content and at absorbance 330 nm and 420 nm of the
protein extract solutions were determined and the results appear in
the following Table XXXVIII:
TABLE-US-00054 TABLE XXXVIII A330 A420 % Protein Control Ext. 97.8
2.84 1.92 Room temperature 78.4 2.57 2.01 40.degree. C. 60.1 1.97
2.23 45.degree. C. 58.2 2.29 1.79
As may be seen from this Table, protein loss can be avoided while
approximately 40% of the phenolic compounds and 30% of the A420
absorbing material are removed by pre-extracting the meal with
ethanol at 40.degree. C.
Example 12
This Example illustrates the preparation of a canola protein
isolate with ethanol extraction of meal.
Eleven 600 g aliquots of dehulled oil seed meal were subjected to
four 3 L ethanol extractions using a meal to ethanol w/v ratio of
1:5. The extractions were done for 30 minutes at 35.degree. C.
Following the 30 minute mixing time, the slurry was allowed to
settle and the supernatant was poured off. UV absorbances at A330
and A420 were determined for each extraction and a protein content
measurement was carried out on the first extraction from the first
aliquot of meal extracted.
Following the fourth extraction, the meal was spread out in a
shallow pan in a fume hood and allowed to dry overnight. The entire
batch of washed meal was allowed to desolventize in the fume hood
for one more night before the 5.4 kg of dried extracted meal was
used in a 50 L batch extraction.
With each extraction of the sample, the colour of the supernatant
became progressively lighter and A330 and A420 decreased. On
average, a 5-fold reduction in A330 and a 6-fold reduction in A420
was seen. The absorbance values are A420 and A330 respectively for
the various extracts are set forth in the following Tables XXXIX
and XL:
TABLE-US-00055 TABLE XXXIX Absorbance at A420 Meal Aliquot Extract
1 Extract 2 Extract 3 Extract 4 A 1.688 0.606 0.276 0.181 B 0.87
0.379 0.124 0.103 C 0.891 0.432 0.206 0.129 D 0.182 0.334 0.218
0.116 E 0.8 0.366 0.159 0.093 F 0.896 0.469 0.215 0.114 G 0.8 0.398
0.194 0.122 H 0.821 0.376 0.203 0.117 I 0.836 0.398 0.189 0.111 J
0.827 0.375 0.203 0.131 K 0.833 0.402 0.265 0.115
TABLE-US-00056 TABLE XL Absorbance at A330 Meal Aliquot Extract 1
Extract 2 Extract 3 Extract 4 A 98.1 47.5 22.1 12.8 B 30.7 14.7
13.9 10.43 C 61.2 27.8 15.3 10.9 D 57.5 25.3 19.5 11.37 E 60.4 27.7
16.2 10.4 F 58.6 29.3 18.4 11.4 G 58.8 28.6 17.3 12 H 57.9 26.3
14.3 10.02 I 60.1 29.3 15.6 11.02 J 56.7 30.1 17.8 10.89 K 61.2
27.98 14.77 11.08
The 5 kg of ethanol-extracted meal was added to 50 L of 0.15 M NaCl
and mixed for 30 minutes at a room temperature of 20.degree. C.
with 0.05 wt % ascorbic acid added to the slurry after 15 minutes
as an antioxidant.
The residual canola meal was removed and washed on a vacuum filter
belt. The resulting protein solution was clarified by filtration
through a 20 .mu.m bag filter followed by centrifugation at 6500
rpm for 5 minutes to produce 39.6 L of protein solution having a
protein content of 23.7 g/L.
37.55 L of the clarified protein solution was concentrated to 3 L
using an ultrafiltration system using 10,000 dalton molecular
weight cut-off membranes. The concentrated protein solution was
diafiltered on a diafiltration system using 10,000 dalton molecular
weight cut-off membranes using 24 L (=8 retentate volumes) of 0.15
M NaCl containing 0.05 wt % ascorbic acid. The resulting 3L of
concentrated and diafiltered canola protein solution had a protein
content of 184 g/L.
The concentrated and diafiltered solution at 30.degree. C. was
diluted into 30 L of water having a temperature of 4.degree. C. A
white cloud of protein micelles formed immediately and was allowed
to settle. The supernatant was removed and the precipitated,
viscous, sticky mass (PMM) (5.78 kg) was removed from the bottom of
the vessel and spray dried. The dried protein isolate had a protein
content of 101.2 wt % (N.times.6.25) d.b.
26 L of supernatant from the micelle formation was concentrated to
3 L by concentration on an ultrafiltration system using 10,000
dalton molecular weight cut-off membranes. The concentrated
supernatant then was diafiltered on a diafiltration system using
10,000 dalton molecular weight cut-off membranes with 6 L of
water.
The concentrated and diafiltered solution was dried and the dried
protein was found to have a protein content of 101.3 wt %
(N.times.6.25) d.b.
Samples of the PMM-derived canola protein isolate (CPI) and the
supernatant derived canola protein isolate were analyzed for
lightness (L) and chromaticity (a and b) using a Minolta R-310
colorimeter. The following Table XLI shows the results
obtained:
TABLE-US-00057 TABLE XLI Sample L a b PMM-derived CPI 84.32 -1.84
23.85 Supernatant-derived CPI 81.92 -0.5 14.18
These products were quite light with `a` and `b` values suggesting
relatively lower levels of red and yellow colour rotes.
SUMMARY OF DISCLOSURE
In summary of this disclosure, the present invention provides with
the recovery of canola protein isolates having decreased colour, by
effecting one or more operations during isolate preparation
designed to remove colourant-causing components, inhibition of
oxidation of colourant-causing components and the removal of
colourants. Modifications are possible within the scope of the
invention.
* * * * *